Systems and methods of fluidic sample processing

ABSTRACT

The present invention provides fluidic devices and systems that allow detection of analytes from a biological fluid. The methods and devices are particularly useful for providing point-of-care testing for a variety of medical applications.

CROSS REFERENCE

This application claims priority to U.S. Provisional Application No.60/954,301 filed on Aug. 6, 2007, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

The discovery of a vast number of disease biomarkers and theestablishment of miniaturized microfluidic systems have opened up newavenues to devise methods and systems for the prediction, diagnosis andtreatment of diseases in a point-of-care setting. Point-of-care testingis particularly desirable because it rapidly delivers results to medicalpractitioners and enables faster consultation. Early diagnosis allows apractitioner to begin treatment sooner and thus avoiding unattendeddeterioration of a patient's condition.

In many diagnostic devices that perform immunoassays, dilution of thetest sample prior to running the assay is preferable for a variety ofreasons. For example, sample dilution can help removal of matrixeffects, unbind the target molecules from other binding proteins in thesample, bring the target analyte concentration into the measurable rangeof the assay, and/or provide sufficient liquid to perform multipleassays (especially when the available sample is small, such as a drop offluid). In most cases, the dilution must be exquisitely precise or riskcompromising the precision of the assay result. Devices such as thoseconstructed by Abaxis (U.S. Pat. No. 5,472,603), Biotrack (U.S. Pat.Nos. 4,946,795 and 5,104,813), and Miles (U.S. Pat. No. 5,162,237)typically perform dilutions with a precision to about 2%; however, suchdevices may require a large volume of test sample, which is moredifficult to obtain in a point-of-care setting.

Often, when measuring a blood sample, analytes in serum or plasma are ofinterest. One reason is that many analytes are found in the fluid partof blood namely plasma. For numerous blood tests performed for clinicalpurposes, the final reported concentration typically needs to relate tothe concentration of blood serum or blood plasma in a diluted sample. Inmost cases, blood serum or blood plasma is the test medium of choice inthe lab. Two operations may be necessary prior to running an assay,dilution and red blood cell removal. Blood samples vary significantly inthe proportion of the sample volume occupied by red cells (thehematocrit which varies from about 20-60%). Furthermore, in apoint-of-care environment when assay systems are operated by non-expertpersonnel, the volume of sample obtained may not be that which isintended. If a change in volume is not recognized, it can lead to errorin the reported analyte concentrations.

Thus, there remains a considerable need for point-of-care devices thatcan provide accurate and rapid data collection, transmission, analysis,and/or real-time medical consultation or decision making. In particular,there remains a need in the art for a point-of-care fluidic device thatcan measure the concentration of plasma in a sample, and determined theapparent dilution ratio to effect an accurate quantification of ananalyte of interest present in a small sample of blood. The presentinvention satisfies these needs and provides related advantages as well.

SUMMARY OF THE INVENTION

In an aspect of the invention, a fluidic device for detecting thepresence or absence of an analyte in a bodily fluid from a subjectcomprises a sample collection unit, an assay assembly, and a calibrationunit, wherein the sample collection unit contains a diluent and isremoveable from the device. In an embodiment, the sample collection unitis configured to collect a sample of bodily fluid from a subject. In anembodiment, the assay assembly comprises at least one reaction sitecontaining a reactant that reacts with an analyte to yield a detectablesignal indicative of the presence of the analyte.

In another embodiment, a device comprises a calibration unit, whereinthe calibration unit is configured to provide a measurement of a sampleused for calibrating a detectable signal.

In a further aspect of the invention, a fluidic device is disclosed fordetecting the presence or absence of an analyte in a bodily fluid from asubject comprising a sample collection unit, an assay assembly, and acalibration unit. The sample collection unit is configured to collectsaid sample of bodily fluid from said subject. The assay assemblycomprises at least one reaction site containing a reactant that reactswith the analyte to yield a detectable signal indicative of the presenceof the analyte. The calibration unit is configured to provide ameasurement of said sample used for calibrating said detectable signal.

In an embodiment, the measurement of a sample is used for determiningconcentration of the detected analyte.

In another embodiment, a sample collection unit of the device allows asample of bodily fluid to react with a reactant contained within anassay assembly based on a protocol transmitted from an external device.

In some embodiments, a sample of bodily fluid is a blood sample.Preferably, the sample is less than 50 microliters. In an embodiment,the sample is a single drop of fluid.

In an embodiment, the sample collection unit is removeable from thedevice. In another embodiment, the sample collection unit is furtherconfigured to dilute a sample. The sample collection unit can contain adiluent for diluting a sample.

In another embodiment, the sample collection unit is further configuredto remove a part of a sample. For example, the unit can remove cellsfrom a sample such as blood.

In an embodiment, a reaction site within an assay assembly isimmobilized in a fluidic channel. In an embodiment of the device, thereaction site receives reagents from a first direction and a sample ofbodily fluid from a second direction. The reagents and the sample can bedelivered to the reaction site by syringe action.

In an embodiment, the device conducts an immunoassay. The reagents forcarrying out the reaction comprise immunoassay reagents. In anembodiment, the detectable signal yielded at a reaction site is aluminescent signal. The device can also be configured to detect aplurality of analytes useful for assessing efficacy and/or toxicity of atherapeutic agent.

In another embodiment, a calibration unit of the device is configured tomeasure the conductivity of a sample. To measure the conductivity of asample, the device can comprise electrodes. In a preferable embodiment,the electrodes are positioned within a fluidic channel.

In an embodiment, the sample collection unit, assay assembly, andcalibration unit can also be configured for one time use.

In an embodiment, the device of the invention further comprises a wastechamber.

In another aspect of the invention, a system for detecting an analyte ina bodily fluid from a subject is disclosed. The system comprises afluidic device of the invention, a reader assembly comprising adetection assembly for detecting a signal, and a communication assemblyfor transmitting the signal to an external device.

In an embodiment, a sample collection unit of the system of theinvention allows a sample of bodily fluid to react with reactantscontained within an assay assembly based on a protocol transmitted froman external device to yield a detectable signal indicative of thepresence of an analyte. In a further embodiment, the protocol istransmitted wirelessly from an external device. In another embodiment, afluidic device further comprises an identifier to provide the identityof said fluidic device that is adapted to trigger the transmission of aprotocol.

In an embodiment, a system of the invention is configured to detect aplurality of analytes useful for assessing efficacy and/or toxicity of atherapeutic agent.

In an aspect of the invention, a method of detecting an analyte in abodily fluid from a subject is disclosed, which comprises providing afluidic device or system of the invention, allowing a portion of asample to react with assay reagents contained within an assay assemblyof the device to yield a signal indicative of the presence of an analytein the sample, and detecting the signal generated from the analytecollected in the sample of bodily fluid.

In an embodiment, a method of the invention comprises calibrating themeasurement of a signal generated from an analyte in a sample based upona measurement by a calibration unit of an embodiment of a device of theinvention. In a further embodiment, an additional step of quantifyingthe amount of an analyte present in a bodily fluid based on themeasurement provided by the calibration unit can be executed.

In an embodiment, a fluidic device can detect a plurality of analytesand the fluidic device comprises immunoassay reagents for the pluralityof analytes.

In an aspect of the invention a method of measuring plasma concentrationof a blood sample comprises providing a plasma sample substantially freeof removing red blood cells, passing a current through the plasmasample, and measuring at least one of conductivity or impedance of theplasma sample, thereby measuring the plasma concentration of said bloodsample.

In an embodiment, the blood sample is diluted with a diluent prior topassing a current through the plasma sample.

In an embodiment, measuring of plasma concentration occurs in a fluidicchannel.

In another embodiment, a method of measuring plasma concentration of ablood sample comprises comparing the measured conductivity to a set ofpredetermined values showing relationship of conductivity values andplasma concentrations.

In an embodiment, a method of measuring plasma concentration of a bloodsample comprises comparing the measured conductivity to a set ofpredetermined values showing relationship of conductivity values anddilution ratios that are employed to dilute the blood sample. In furtherembodiment, the set of predetermined values is a calibration curve. Thecalibration curve can be normalized. In some embodiments, normalizingaccounts for at least one factor selected from the group consisting ofsurface area of an electrode, temperature of the sample, gain of acircuit wherein the circuit delivers the current to the sample, volumeof the sample, and red blood cell quantity of the sample.

In an aspect, a method of calculating an apparent dilution ratioutilized in diluting a blood sample for running a blood test comprisesproviding a plasma sample derived from a diluted blood sample, measuringconductivity of the plasma sample, and comparing the measuredconductivity to a set of predetermined values showing relationship ofconductivity values and dilution ratios that are employed to dilute ablood sample, thereby calculating the apparent dilution ratio.

In an embodiment of the method of the invention, the blood sample isdiluted prior to running the blood test.

In another embodiment, the plasma sample derived from a diluted bloodsample is substantially free of red blood cells.

In an embodiment, the conductivity of the plasma sample is inverselyproportional to the apparent dilution ratio.

The present invention also provides a business method of monitoring aclinical trial of a therapeutic agent, comprising: a) collecting atleast one pharmacological parameter from a subject in said clinicaltrial at a plurality of time intervals, said collecting step is effectedat each time interval by subjecting a sample of bodily fluid from saidsubject to reactants contained in a subject fluidic device, wherein saidfluidic device is provided to said subject to yield detectable signalsindicative of the values of said at least one pharmacological parameterat a plurality of time intervals; b) comparing the detected values to athreshold value predetermined for said pharmacological parameter; c)notifying a clinician and/or a sponsor involved in said clinical trialwhen a statistically significant discrepancy exists between the detectedvalues and the threshold value. In one embodiment, the business methodinvolves the step of taking a medical action based on the statisticallysignificant discrepancy. Such method action can involve adjusting dosageof the therapeutic agent, continuing, modifying, or terminating theclinical trial.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates a device for fluidic sample processing.

FIG. 2 illustrates the components of a device for fluidic sampleprocessing and demonstrates some relationships between the components.

FIG. 3 illustrates a sample collection unit containing a diluent.

FIG. 4 illustrates a sample dilution syringe for collecting and dilutinga sample with a diluent.

FIG. 5 illustrates a part of a device for fluidic sample processingcomprising a cartridge, a liquid routing manifold, and a samplecollection well.

FIG. 6 illustrates a liquid routing manifold for routing reagents to acartridge for sample processing.

FIG. 7 illustrates a vial for containing reagents for fluidic sampleprocessing and demonstrates the assembly of the vial.

FIG. 8 illustrates a reagent container formed by blow molding.

FIG. 9 demonstrates a cartridge for sample processing.

FIG. 10 demonstrates a circuit block diagram for measuring electricalconductivity of a fluidic sample.

FIG. 11 demonstrates a microfluidic channel adjacent to electrodes thatcan be incorporated in a sample processing cartridge for measuringelectronic conductivity of a fluidic sample.

FIG. 12 demonstrates an exemplary chart comparing the concentration ofplasma in a plasma dilution to the reference measurement of the plasmadilution fluidic sample.

FIG. 13 demonstrates the measurable range of concentration toconductivity signal and the effect of changing the gain setting on thecircuit.

FIG. 14 demonstrates effect of the distance between electrodes adjacentto a microfluidic channel on the measurement of plasma concentration.

FIG. 15 illustrates of a plot of the surface area of an electrode as itrelates to the voltage measured by the circuit.

FIG. 16 demonstrates the effect of a change in volume of the plasmadilution sample on the signal measured to determine the conductivity ofthe sample.

FIG. 17 demonstrates that the temperature of fluidic sample can effectthe conductivity measurement.

FIG. 18 illustrates a method of normalizing the conductivity measurementof the plasma concentration by measuring the conductivity of a referencebuffer.

FIG. 19 demonstrates the result of a method of normalizing theconductivity measurement of the plasma concentration according totemperature of the fluidic sample.

FIG. 20 demonstrates the effect of the amount of red blood cells in theplasma dilution sample on the measured conductivity of the solution.

FIG. 21 demonstrates the effect of the amount of lysed red blood cellsin the plasma dilution sample on the measured conductivity of thesolution.

FIG. 22 illustrates the result of the measuring the plasma dilutionratio of four subjects and demonstrates a comparison to a standardcalibration line.

FIG. 23 illustrates an exemplary assay response for detecting Protein Cin a blood sample using a fluidic processing device of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Fluidic Devices and Systems

One aspect of the present invention is a system for detecting an analytein a sample of bodily fluid. The system is capable of detecting and/orquantifying analytes that are associated with specific biologicalprocesses, physiological conditions, disorders or stages of disorders,or effects of biological or therapeutic agents.

In an aspect, a fluidic device for detecting the presence or absence ofan analyte in a bodily fluid from a subject comprises a samplecollection unit and an assay assembly, wherein the sample collectionunit contains a diluent and is removable from the device. In anembodiment, the sample collection unit is configured to collect a sampleof bodily fluid from a subject. In an embodiment, the assay assemblycomprises at least one reaction site containing a reactant that reactswith an analyte to yield a detectable signal indicative of the presenceof the analyte.

In another embodiment, a device comprises a calibration unit, whereinthe calibration unit is configured to provide a measurement of a sampleused for calibrating a detectable signal.

The system comprises a fluidic device having one or more of thefollowing components: a sample collection unit, an assay assembly, and acalibration unit. The sample collection unit is configured to collect asample of bodily fluid from a subject. The assay assembly comprises atleast one reaction site containing a reactant that reacts with ananalyte from the bodily fluid, which yields a detectable signalindicative of the presence of the analyte.

In general, the calibration unit is configured to provide a measurementof the sample used for calibrating the detected signal. For example, thecalibration unit can be any unit or device part that measures aparameter for calibrating the measurement of the concentration of ananalyte in a sample. Exemplary calibration units can provide withoutlimitation volume measurement, flow measurement, and cell count. In oneaspect, measurement provided by the calibration unit is used fordetermining the concentration of the detected analyte. For example,where a diluted blood sample is used for detecting analyte present inthe plasma portion, the calibration unit provides a measurement of thediluted plasma concentration, and hence the apparent dilution ratiowhich is then in turn used for determining the initial concentration ofthe analyte of interest.

Any bodily fluids suspected to contain an analyte of interest can beused in conjunction with the system or devices of the invention.Commonly employed bodily fluids include but are not limited to blood,serum, saliva, urine, gastric and digestive fluid, tears, stool, semen,vaginal fluid, interstitial fluids derived from tumorous tissue, andcerebrospinal fluid.

A bodily fluid may be drawn from a patient and brought into the fluidicdevice in a variety of ways, including but not limited to, lancing,injection, or pipetting. In one embodiment, a lancet punctures the skinand draws the sample into the fluidic device using, for example,gravity, capillary action, aspiration, or vacuum force. The lancet maybe part of the fluidic device, or part of a reader assembly, or as astand alone component. Where needed, the lancet may be activated by avariety of mechanical, electrical, electromechanical, or any other knownactivation mechanism or any combination of such methods. In anotherembodiment where no active mechanism is required, a patient can simplyprovide a bodily fluid to the fluidic device, as for example, couldoccur with a saliva sample. The collected fluid can be placed in thesample collection unit within the fluidic device. In yet anotherembodiment, the fluidic device comprises at least one microneedle whichpunctures the skin. The microneedle can be used with a fluidic devicealone, or can puncture the skin after the fluidic device is insertedinto a reader assembly.

The volume of bodily fluid to be used with a fluidic device of thepresent invention is generally less than about 500 microliters,typically between about 1 to 100 microliters. Where desired, a sample of1 to 50 microliters, 1 to 40 microliters, 1 to 30 microliters, 1 to 10microliters or even 1 to 3 microliters can be used for detecting ananalyte using the subject fluidic device.

In an embodiment, the volume of bodily fluid used for detecting ananalyte utilizing the subject devices or methods is one drop of fluid.For example, one drop of blood from a pricked finger can provide thesample of bodily fluid to be analyzed with a fluidic device system ormethod of the invention.

In some embodiments, the bodily fluids are used directly for detectingthe analytes present therein with the subject fluidic device withoutfurther processing. Where desired, however, the bodily fluids can bepre-treated before performing the analysis with the subject fluidicdevices. The choice of pre-treatments will depend on the type of bodilyfluid used and/or the nature of the analyte under investigation. Forinstance, where the analyte is present at low level in a sample ofbodily fluid, the sample can be concentrated via any conventional meansto enrich the analyte. Methods of concentrating an analyte include butare not limited to drying, evaporation, centrifugation, sedimentation,precipitation, and amplification. Where the analyte is a nucleic acid,it can be extracted using various lytic enzymes or chemical solutionsaccording to the procedures set forth in Sambrook et al. (“MolecularCloning: A Laboratory Manual”), or using nucleic acid binding resinsfollowing the accompanying instructions provided by manufacturers. Wherethe analyte is a molecule present on or within a cell, extraction can beperformed using lysing agents including but not limited to denaturingdetergent such as SDS or non-denaturing detergent such as Thesit, sodiumdeoxylate, triton X-100, and tween-20.

In an embodiment, pretreatment can include diluting and/or mixing thesample. In addition, the pretreatment can include filtering the sampleto remove, for example, red blood cells from a blood sample. In oneaspect, the subject systems and fluidic devices are provided with thecapability of on-board sample pretreatment, including without limitationdilution, concentration, and separation of a particular sample fraction(for example, separating bodily fluid from cells contained therein).

In an embodiment, the sample is diluted in a ratio that is satisfactoryfor a both a high sensitivity and low sensitivity assays. For example, adilution ratio of sample to diluent can be in the range of about1:100-1:1. In another example, the dilution ratio is about 1:15.

In an embodiment, the device of the invention is self-contained andcontains all reagents (liquid and solid-phase) required to performseveral quantitative immunoassays in a short period of time. The devicecan use a small volume of bodily fluid, for example, a single drop. Inan embodiment, the bodily fluid is blood and can be obtained by afingerstick. The device can be operated by a small instrument. Differentfunctions of that can be carried out by the device include, but are notlimited to, dilution of a sample, removal of parts of a sample (forexample, red blood cells (RBCs), fluidic routing of a sample to an assayassembly, routing of the liquid reagents to the assay assembly, andcontaining liquids during and following use of the device. The devicecan also include an optical interface.

In some embodiments, the sample collection unit is configured to collecta sample of bodily fluid from the subject and to deliver a predeterminedportion of the sample to be assayed by the assay assembly. In thismanner, the device automatically meters the appropriate volume of thesample that is to be assayed. In an embodiment, the sample collectionunit can comprise a sample collection well and/or a sample dilutionsyringe. Generally, the sample dilution syringe collects bodily fluidfrom the patient.

FIG. 1 illustrates an exemplary system of the present invention. In theembodiment illustrated in FIG. 1, a sample dilution syringe (SDS) isinserted into a fully assembled device. The fluidic device of theinvention may take a variety of configurations, including a single-useunit. In an exemplary system, the device contains the reactantsnecessary to perform one or more assays in a plurality of vials withinthe device. The vials can contain one or more reactants, a wash buffer,or a combination thereof. In other examples, reagents with a relativelyshort shelf-life can be supplied separately and be utilized, forexample, by the end user.

A sample collection unit in a fluidic device may provide a bodily fluidsample from a patient by any of the methods described above. Wheredesired, the sample may first be processed by diluting the bodily fluid,and or may be filtered by separating the plasma from the red bloodcells. In some embodiments there may be more than one sample collectionunit in the fluidic device.

FIG. 2 illustrates exemplary layers of a fluidic device according to thepresent invention prior to assembly of the fluidic device. The samplecollection unit can be a sample dilution syringe for sample acquisitionand capable of diluting a sample that is inserted into the body of thedevice. The sample dilution syringe can contain a capillary tube, whichpress-fits into the housing of the sample dilution syringe. Thecapillary tube can be made from a variety of materials, includingplastic or a variety of polymeric materials such as polystyrene,polycarbonate, polypropylene, polydimethysiloxanes (PDMS), polyurethane,polyvinylchloride (PVC), and polysulfone, or glass, or semi-conductingmaterial such as silicon and its derivatives. The tube can also beoptionally coated with anti-coagulant. An example of the sample dilutionsyringe is illustrated in FIGS. 3 and 4.

In some embodiments the inner surface of the capillary tube, the sampledilution syringe, and/or the sample collection unit may be coated with asurfactant and/or an anti-coagulant solution. The surfactant provides awettable surface to the hydrophobic layers of the fluidic device andfacilitate filling of the capillary tube and sample dilution syringewith a liquid sample, for example, blood. The anti-coagulant solutionhelps prevent a sample, for example, blood, from clotting when providedto the fluidic device. Exemplary surfactants that can be used includewithout limitation, Tween, Triton, Pluronics and other non-hemolyticdetergents that provide the proper wetting characteristics of asurfactant. EDTA and heparin are non-limiting anti-coagulants that canbe used.

In one embodiment, a coating can be made a solution comprising, forexample, 2% Tween, 25 mg/mL EDTA in 50% Methanol/50% H₂O, which is thenair dried. A methanol/water mixture provides a means of dissolving theEDTA and Tween, and also dries quickly from the surface of the material.The solution can be applied to the layers of the fluidic device by anymeans that will ensure an even film over the surfaces to be coated, suchas pipetting, spraying, printing, or wicking.

The housing of the sample dilution syringe comprises a tube. In thetube, two moveable seals can contain a volume of a diluent. In anembodiment, the volume of the diluent is predetermined, for example, inabout the range of 50 microliters to 1 ml, preferably in the range ofabout 100 microliters to 500 microliters.

The diluent is preferably a buffered solution containing anti-coagulant,sugar, antibodies to a common red cell antigen. The diluent can alsocontain a dispersion of magnetizable particles to which are boundantibodies typically directed to red cell antigen. The housing can alsocontain a mixing ball that can aid in re-suspending the magnetizableparticles.

In some embodiments the housing includes a movable mixing element thatcauses the mixing of the predetermined portion of the sample with thediluent. Exemplary moveable mixing element is with a general ball shape.The movable mixing elements may have any shape, such as, for example, acube shape.

In one embodiment the movable mixing element is magnetically controlled,for example, a magnetically controlled ball in the mixing chamber that,when magnetically controlled, will cause the mixing of the predeterminedportion of the sample and the diluent. The ball can be about 5% of thecombined volume of the sample and diluent. The ball can be magneticallycontrolled to move in a reciprocal, linear fashion, within the mixingchamber. The ball may be pre-magnetized or unmagnetized, but susceptibleto magnetic forces.

The moveable mixing element can be contained in the sample dilutionsyringe or in a sample collection well. It is also contemplated that themixing element might operate outside of the fluidic device, such as ifthe reader assembly were adapted to agitate the fluidic device andthereby mix the predetermined portion of sample and the diluent.

In some embodiments the sample collection unit further comprises afilter configured to filter the diluted sample before it is assayed.

FIG. 2 also illustrates a bottom part covered by the body of anexemplary device and below the sample collection unit. An exemplarybottom part of the device is illustrated in FIG. 5 which contains aclear material bottom, an assay assembly unit, a liquid reagentdistribution manifold, and reagent containers. The clear bottom part cancomprise energy directors, which enable ultrasonic welding to the assayassembly unit. When the two parts are welded together, they can define afluid path. In a preferable embodiment, the fluid path comprises acalibration unit, followed by at least one assay assembly. In anembodiment, a plurality of assay assemblies (two, three, four, five,six, or more) is incorporated into the device. Multiple assay assembliespreferably constitute optically and chemically isolated zones.

Also illustrated in FIG. 5 is a cartridge body with features that form afluid channel connecting a sample collection well that is designed tohold a diluted sample to an assay assembly. Where desired, the cartridgecan be made of substantially opaque material. In a preferableembodiment, the opaque cartridge body is constructed of a whitematerial, for example, white plastic. The features of the cartridge caninclude a Greek letter Psi-shaped channel and “vias” (through holes)that are in fluid communication with reagent reservoirs.

At least one of the fluidic channels will typically have small crosssectional dimensions. In some embodiments the dimensions are from about0.01 mm to about 5 mm, preferably from about 0.03 mm to about 3 mm, andmore preferably from about 0.05 mm to about 2 mm. Fluidic channels inthe fluidic device may be created by, for example without limitation,precision injection molding, laser etching, embossing or any othertechnique known in the art to carry out the intent of the invention.

Reagent reservoirs contained within a device of the invention can becoupled to the assay assembly for delivering the reagents to theassembly. One method of coupling the reagent reservoirs and the assayassembly is by utilizing a distribution manifold. FIG. 6 demonstrates anembodiment of a liquid reagent distribution manifold fitted with a setof needles that engage with reagent reservoirs. The manifold comprisesneedles or pins for coupling to a vial containing a reagent.

FIG. 2 illustrates a set of vertically oriented cylindrical reagentreservoirs or vials. In an embodiment, the reagent reservoirs areconstructed of glass. The vials can be fitted with one upper moveableseal and a lower septum. The lower septum can be held in place by acrimpable metal cap. Liquid reagents can be contained the seal and thelower septum. In one version of the invention, during manufacture of thedevice, the lower septum of each reagent reservoir is pierced thecorresponding needles without the needles penetrating into the liquidcompartments. An example of a vial useful for constructing the device ofthe invention is illustrated in FIG. 7.

In a preferred embodiment there is at least one reagent reservoir. Insome embodiments there is any number of reagent reservoirs as arenecessary to fulfill the purposes of the invention. A reagent reservoiris preferably in fluid communication with at least one reaction site,and the reagents contained in the reagent reservoirs can be releasedinto the fluidic channels within the fluidic device.

In an embodiment, the reagent reservoir contains a plurality ofreagents. The plurality of reagents can be separated from each other, toavoid interaction between the reagents. In a further embodiment, areagent reservoir contains a plurality of moveable seals. For example,there may be four moveable seals. Three voids can be created between themoveable seals and the voids can be filled with a reagent. The seals canbe moved by pump or syringe-like action. If there is a hole, forexample, in the side of the reagent reservoir, each void can be movedinto position with the hole, and a reagent contained within the void isthen released through the hole to the reaction site. As such, thereagent reservoir can contain all of the agents or wash buffersnecessary for a single assay.

Reagents according to the present invention include without limitationwash buffers, enzyme substrates, dilution buffers, conjugates,enzyme-labeled conjugates, DNA amplifiers, sample diluents, washsolutions, sample pre-treatment reagents including additives such asdetergents, polymers, chelating agents, albumin-binding reagents, enzymeinhibitors, enzymes, anticoagulants, red-cell agglutinating agents,antibodies, or other materials necessary to run an assay on a fluidicdevice. An enzyme conjugate can be either a polyclonal antibody ormonoclonal antibody labeled with an enzyme that can yield a detectablesignal upon reaction with an appropriate substrate. Non-limitingexamples of such enzymes are alkaline phosphatase and horseradishperoxidase. In some embodiments the reagents comprise immunoassayreagents. In general, reagents especially those that are relativelyunstable when mixed with liquid are confined in a defined region (forexample, a reagent reservoir) within the subject fluidic device. Thecontainment of reagents can be effected by valves that are normallyclosed and designed for one-time opening, preferably in a unidirectionalmanner.

In some embodiments a reagent reservoir contains approximately about 10μl to about 1 ml of fluid. In some embodiments the chamber may containabout 100-500 μl of fluid. The volume of liquid in a reagent chamber mayvary depending on the type of assay being run or the sample of bodilyfluid provided. In an embodiment, the volumes of the reagents do nothave to predetermined, but must be more than a known minimum. In someembodiments the reagents are initially stored dry and dissolved uponinitiation of the assay being run on the fluidic device.

In an embodiment, the reagent reservoir can be filled using a syringe, aneedle, or a combination thereof. The reagent reservoirs may be filledwith fluid using a fill channel and a vacuum draw channel.

In an embodiment of the invention, multiple vials are utilized toisolate reagents from each other. The vials may also be used to containa wash solution. In addition, the vials may be used to contain aluminogenic substrate.

Another embodiment can feature a reagent reservoir vial, a wash solutionvial, and a luminogenic substrate vial all in relation to the same assayassembly.

Other configurations of reservoirs are contemplated such ascollapse-able (concertina-like) plastic vials made of, for example,polypropylene or polyethylene. An example of the collapse-able plasticvials is illustrated in FIG. 8.

The cartridge of the device as shown in FIG. 2 comprises an assayassembly. The assay assembly comprises a “capture surface” or reactionsite, may be placed on any of several locations within the cartridge.Another view of the cartridge is illustrated in FIG. 9. The assayassembly can be located in the “vias” of the cartridge. In anembodiment, the assay assembly is located on the clear cartridge bottom.In a preferable embodiment, the assay assembly is located on or in afluidic channel feature of the cartridge body.

The reaction site can be bound covalently or by adsorption antibodies toan area of the device. The surface is then dried and maintained in drycondition until used in an assay. In an embodiment, there is onereaction site for each analyte to be measured.

In preferred embodiments the reagent reservoirs and sample collectionunit are fluidly connected to reaction sites where bound probes candetect an analyte of interest in the bodily fluid sample using theassay. A reaction site could then provide a signal indicative of thepresence of the analyte of interest, which can then be detected by adetection device described in detail herein below.

In an alternative embodiment, the capture surface can be located in afluidic channel. In an embodiment, the reaction site can be located inthree, horizontally oriented, fluidic channels. The reaction site can beimmobilized to either the clear bottom surface and/or the cartridgebody. In another embodiment, the reaction site can be located in thevias of a device of the invention. The vias are fluidly connected to thefluidic channel and to the reagent manifold.

In an embodiment, a reaction site can be on a channel that was madeseparately and inserted into or onto the device. One version of thisapproach utilizes molded polystyrene surfaces with the inner surfacecoated with a reaction site, wherein the surfaces can be fitted into oradjacent to the channels or vias of the device. Similarly, a reactionsite can be coated onto the inside of cylindrical polystyrene tubeswhich are press fitted into a fluid channel or fitted into a via. Thesemolded surfaces can provide an assay capture surface that can be madeand evaluated before the assembly of the cartridge for bulkmanufacturing for one-time use and replacement of the reaction site. Inanother embodiment, a molded plastic piece can be placed in the deviceof the present disclosure by a variety of methods. For example, it canbe compressed into a fluid channel. In another example, the reactionsite can be fit into a cavity in a fluidic channel with inlets andoutlets smaller than the reaction site (for example, a cavity is formedwhen two parts of the disposable are welded together). Some advantagesof this embodiment include, but are not limited to, eliminating pressfit methods; and making large quantities of the reaction site asmentioned previously. The reaction site may be quality controlled beforeassembly of the overall cartridge.

In another alternative embodiment, an object can be placed into a via orfluid channel of the device. The object within a via or fluid channelcan be coated with a capture surface, creating a reaction site of theinvention. The object can be placed in the vias or channels of thedevice before, during, or after device construction and incorporatingthe reaction site into the device. In one embodiment, molded plastic(such as polystyrene) pieces such as spheres, beads, elliptical forms,torroidal elements, and the like are either commercially available orcan be made by injection molding with precise shapes and sizes. Forexample, the characteristic dimension can be in the 0.05-3 mm range.These pieces can be coated with capture reagents using a method similarto those used to coat microtiter plates but with the advantage that theycan be processed in bulk by placing them in a large vessel, addingcoating reagents and processing using sieves and the like to recover thepieces and wash them as needed. In this way, a large number of reactionsites can be manufactured on the vias or the channels of themicrofluidic device for mass production of the devices.

In another embodiment, an area of the device comprises projections thatare coated with capture reagents to carry out an assay with the deviceof the invention.

In some embodiments the reactions sites are flat but they may take on avariety of alternative surface configurations. The reaction sitepreferably forms a rigid support on which a reactant can be immobilized.The reaction site surface is also chosen to provide appropriatecharacteristics with respect to interactions with light. For instance,the reaction site may be functionalized glass, Si, Ge, GaAs, GaP, SiO₂,SiN₄, modified silicon, or any one of a wide variety of gels or polymerssuch as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride,polystyrene, polycarbonate, polypropylene, or combinations thereof.Other appropriate materials may be used in accordance with the presentinvention. A transparent reaction site may be advantageous. In addition,in the case where there is an optically transmissive ‘window” permittinglight to reach an optical detector, the surface may be advantageouslyopaque and preferentially light scattering.

A reactant immobilized at a reaction site can be anything useful fordetecting an analyte of interest in a sample of bodily fluid. Forinstance, such reactants include without limitation nucleic acid probes,antibodies, cell membrane receptors, monoclonal antibodies and antiserareactive with a specific analyte. Various commercially availablereactants such as a host of polyclonal and monoclonal antibodiesspecifically developed for specific analytes can be used.

One skilled in the art will appreciate that there are many ways ofimmobilizing various reactants onto a support where reaction can takeplace. The immobilization may be covalent or non-covalent, via a linkermoiety, or tethering them to an immobilized moiety. These methods arewell known in the field of solid phase synthesis and micro-arrays (Beieret al., Nucleic Acids Res. 27:1970-1-977 (1999). Non-limiting exemplarybinding moieties for attaching either nucleic acids or proteinaceousmolecules such as antibodies to a solid support include streptavidin oravidin/biotin linkages, carbamate linkages, ester linkages, amide,thiolester, (N)-functionalized thiourea, functionalized maleimide,amino, disulfide, amide, hydrazone linkages, and among others. Inaddition, a silyl moiety can be attached to a nucleic acid directly to asubstrate such as glass using methods known in the art. Surfaceimmobilization can also be achieved via a Poly-L Lysine tether, whichprovides a charge-charge coupling to the surface.

In some embodiments there are more than one reaction sites which canallow for detection of multiple analytes of interest from the samesample of bodily fluid. In some embodiments there are 2, 3, 4, 5, 6, ormore reaction sites, or any other number of reaction sites as may benecessary to carry out the intent of the invention.

In embodiments with multiple reaction sites on a fluidic device, eachreaction site may be immobilized with a reactant different from areactant on a different reaction site. In a fluidic device with, forexample, three reaction sites, there may be three different probes, eachbound to a different reaction site to bind to three different analytesof interest in the sample. In some embodiments there may be differentreactants bound to a single reaction site if, for example, a CCD withmultiple detection areas were used as the detection device, such thatmultiple different analytes could be detected in a single reaction site.The capability to use multiple reaction sites in addition to multipledifferent probes on each reaction site enables multiple analytemeasurement characteristics of the present invention.

The present invention allows for the detection of multiple analytes onthe same fluidic device. If assays with different luminescentintensities are run in adjacent reaction sites, photons (signals thatemanate from the reactions) may travel from one reaction site to anadjacent reaction site, as reaction sites may be constructed ofmaterials that allow photons to travel through the fluidic channels thatconnect the sites. This optical cross talk may compromise the accuracyof the detected photons. Different embodiments of this invention caneliminate or reduce the amount of optical cross-talk. Non-linearchannels prevent photons to pass through. Additionally, the edges orwalls of a reaction site may be constructed using optically opaque orlight scattering materials so that light will not escape. In someembodiments the reaction sites are white or opaque.

In some embodiments, unbound signal-generating conjugates may need to bewashed from a reaction site to prevent unbound conjugates fromactivating the substrate and producing and inaccurate signal. It may bedifficult to remove conjugates sticking to the edges of the reactionsites in such a fluidic device if, for example, there is not an excessof a wash solution. To decrease the signal contributed from unboundconjugates stuck to the edge of a reaction site, it may be advantageousto expand the reaction site edge or wall radius in order to distancenon-specifically bound conjugate from the desired actual detection area,represented by bound reactant.

When using a wash buffer in an assay, the device can store buffer invials in fluid communication with the reaction site. In an embodiment,the wash reagent is able to remove reagent from the reaction sites byabout 99.9% by washing. In general, a high washing efficiency resultingin a high degree of reduction of undesired background signals ispreferred. Washing efficiency is typically defined by the ratio ofsignal from a given assay to the total amount of signal generated by anassay with no wash step and can be readily determined by routineexperimentation. It is generally preferred to increase the volume ofwashing solution and time of incubation but without sacrificing thesignals from a given assay. In some embodiments, washing is performedwith about 200 ul to about 5000 ul of washing buffer, preferably betweenabout 250 ul to about 1000 ul washing buffer, for about 10 to about 300seconds. To facilitate this efficiency, the sides of the reaction sitesare adapted for smooth flow of the reagents and for minimal boundarylayer effects. Where desired, the channels connecting the reaction sitescan be configured as a chicane so as to reduce cross-talk of any kindand also uncontrolled fluid flow.

Additionally, it can be advantageous to use several cycles of smallvolumes of wash solution which are separated by periods of time where nowash solution is used. This sequence allows for diffusive washing, wherelabeled antibodies diffuse over time into the bulk wash solution fromprotected parts of the assay such as the well edges or surfaces where itis loosely bound and can then be removed when the wash solution is movedfrom the reaction site.

Where desired, the subject systems and fluidic devices can be configuredto contain any reagents necessary to perform an assay on a fluidicdevice according to the present invention on-board, or housed within thefluidic device before, during, and after the assay. In this way the onlyinlet or outlet from the fluidic device is preferably the bodily fluidsample initially provided by the fluidic device. This design also helpscreate an easily disposable fluidic device where all fluids or liquidsremain in the device. The on-board design also prevents leakage from thefluidic device into the reader assembly which should remain free fromcontamination from the fluidic device.

In embodiments of the invention the fluidic device includes at least onewaste chamber to trap or capture all liquids after they have been usedin the assay. In an embodiment, there is more than one waste chamber, atleast one of which is to be used with a calibration assembly describedherein below. On-board waste chambers also allow the device to be easilydisposable. The waste chamber is preferably in fluidic communicationwith at least one reaction site.

In addition, a bubble trapper can be positioned between a samplecollection unit and reaction site. The bubble trapper can have suchgeometry that the bubbles tend to migrate towards the edges of thissurface and remain stuck at that service, thereby not entering into thereaction sites.

The size of the channels in the fluidic device can reduce the amount ofair that enters the reaction sites. As the reagent passes through, thewidth of a first channel is about double the width of a channel leadingto the reaction sites. The potential for air to enter into the channelleading to the reaction sites is reduced or eliminated as the air wouldbe forced to squeeze in the smaller channel.

In some embodiments the fluidic device comprises valves that preventbackflow through the circuitry. During assays the volume, velocity, andtiming of the liquid movement can be controlled. Assays may incorporateprolonged incubations where very little, if any, liquid movement shouldoccur. During these stages no or substantially no back-flow of liquidshould occur in the fluidic device.

In certain applications of the subject systems or fluidic devices,calibration of the detected signal is desirable in order to ascertainthe concentration of an analyte present in the initial sample of bodilyfluid under investigation. Accordingly, in a featured embodiment of thepresent invention, the calibration unit can be any unit or device partthat measures a quantity for calibrating the measurement of theconcentration of an analyte in a sample. Example calibration unitsinclude, but are not limited to, a volume measurement, a flowmeasurement, and a cell counter. In a preferable embodiment of theinvention there is a separate control circuit which provides knownquantities of analytes that can be measured in parallel with analytes inthe sample. In another embodiment, the sample is mixed with a knownquantity of a substance not found in a sample, and a measurement of theadded substance is made. By comparison of the known added analytequantity with that measured, the analyte “recovery” can be determinedand used to correct possible errors in measurements of analytes in thesample.

To measure the quantity of plasma in a diluted sample, it is oftendesirable to measure the apparent dilution ratio (sample plasma to finalvolume of diluted plasma). In an embodiment, the conductivity of thediluted sample can be measured to determine the apparent dilution ratio.For measurement of conductivity of a test sample, at least twoelectrodes are typically placed adjacent to a fluidic channel betweenthe sample collection well and the assay assembly. The electrodes may ormay not be in fluid communication with the fluidic channel. In anembodiment, the electrodes are adjacent to the same surface of thefluidic channel. The cartridge illustrated in FIG. 9 demonstrates twoelectrodes in a fluidic channel between a sample collection well and thereaction sites. The electrodes can be spaced at different distances inthe fluidic channel. The spacing and size of the electrodes has aneffect on the measurement of electrical conductivity of a sample asdiscussed later herein. Where desired, the electrodes can be connectedto a control circuit when the methods and systems of the invention areexecuted.

Depending on the intended application, it may be advantageous to measureconductivity in a control as a means to check the measurement system.For example, the control part is exposed to reagents of knownconductivity provided by the manufacturer.

In some embodiments, a sensor for assessing the reliability of an assayfor an analyte in a bodily fluid with the use of the subject fluidicdevice can be provided together with the fluidic device, the readerand/or within the packaging of the subject system. The sensor is capableof detecting a change in operation parameters under which the subjectsystem normally operates. The operation parameters include but are notlimited to temperature, humidity, and pressure, which may affect theperformance of the present system.

A fluidic device and reader assembly may, after manufacturing, beshipped to the end user, together or individually. As a reader assemblyis repeatedly used with multiple fluidic devices, it may be necessary tohave sensors on both the fluidic device and reader assembly to detectsuch changes during shipping, for example. During shipping, pressure ortemperature changes can impact the performance of a number of componentsof the present system, and as such a sensor located on either thefluidic device or reader assembly can relay these changes to, forexample, the external device so that adjustments can be made duringcalibration or during data processing on the external device. Forexample, if the temperature of a fluidic device is changed to a certainlevel during shipping, a sensor located on the fluidic device coulddetect this change and convey this information to the reader assemblywhen it is inserted into the reader assembly by the user. There may bean additional detection device in the reader assembly to perform this,or such a device may be incorporated into another system component. Insome embodiments this information may be wirelessly transmitted toeither the reader assembly or the external device. Likewise, a sensor inthe reader assembly can detect similar changes. In some embodiments, itmay be desirable to have a sensor in the shipping packaging as well,either instead of in the system components or in addition thereto.

Manufacturing of the fluidic channels may generally be carried out byany number of microfabrication techniques that are well known in theart. For example, lithographic techniques are optionally employed infabricating, for example, glass, quartz or silicon substrates, usingmethods well known in the semiconductor manufacturing industries such asphotolithographic etching, plasma etching or wet chemical etching.Alternatively, micromachining methods such as laser drilling,micromilling and the like are optionally employed. Similarly, forpolymeric substrates, well known manufacturing techniques may also beused. These techniques include injection molding or stamp moldingmethods where large numbers of substrates are optionally produced using,for example, rolling stamps to produce large sheets of microscalesubstrates or polymer microcasting techniques where the substrate ispolymerized within a micromachined mold.

In some embodiments at least one of the different layers of the fluidicdevice may be constructed of polymeric substrates. Non limiting examplesof polymeric materials include polystyrene, polycarbonate,polypropylene, polydimethysiloxanes (PDMS), polyurethane,polyvinylchloride (PVC), and polysulfone.

The subject fluidic devices can comprise a waste chamber that containsunreacted reagents. Where desired, the waste chamber can contain anabsorbent material comprises at least one quenching agent which reactswith at least one reagent from said assay assembly to reduceinterference of the optical signal indicative of the presence of theanalyte in the sample. The quenching agent can inhibit the bindingbetween reagents, or in preferred embodiments the quenching agentinactivates at least one and more preferably all reagents which maycontribute to an interfering optical signal.

The reagent or reagents with which the quenching agent in the wastechamber reacts to reduce the interference can be, for example withoutlimitation, an unbound enzyme and/or an unbound substrate. The reagentwith which the quenching agent reacts to reduce the interference isgenerally not as important as the reduction of the interference itself.The quenching agent in the waste chamber can vary depending on the typeof assay that is being performed in the fluidic device. Preferably asubject quenching agent reduces an interfering optical signal by atleast about 95%, at least about 98%, at least about 99%, at least about99.9%, or more. In a preferred embodiment the quenching agent reduces aninterfering optical signal by about 99%. In another preferredembodiments the waste chamber reduces optical interference by at leastabout 99.5%. In more preferred embodiments the quenching agent reducesoptical interference by at least about 99.9%. The quenching effectshould preferably be rapid, typically within a few minutes and morepreferably within a few seconds. It also should preferably be ascomplete as possible to ensure the interference is reduced as much aspossible. In preferred embodiments the inactivation of the enzymereaction should be more than 99% complete before the optical signalindicative of the presence of the analyte in the sample is detected byany detection mechanism that may be used with the fluidic device asdescribed herein.

In some embodiments the quenching agent can be a chemical that is astrong non-volatile acid such as trichloroacetic acid or its salt sodiumtrichloracetate. The substance can also be a strong alkali such assodium hydroxide. Other strong non-volatile acids and strong alkalis canbe used in accordance with the present invention. In some embodimentsthe quenching agent reduces the optical interference by inhibiting theenzyme. In an ELISA, for example, the quenching agent can interfere withthe enzyme's ability to convert the substrate to produce a luminescentsignal. Exemplary enzyme inhibitors include lactose which inhibits theaction of β-galactosidase on luminogenic galactosides, and phosphatesalts which inhibit phosphatases. In other embodiments the quenchingagent can reduce the interference by denaturing the enzyme. Oncedenatured the enzyme it is unable to carry out it enzymatic function andthe optical interference is suppressed or reduced. Exemplary denaturantsinclude detergents such as sodium dodecyl sulfate (SDS), heavy metalsalts such as mercuric acetate, or chelating agents such as EDTA whichcan sequester metal ions essential for activity of certain enzymes suchas alkaline phosphatase. All types of surfactants may be used includingcationic (CTMAB) and anionic (SDS). In a preferable embodiment,azobenzene compounds can be used as a quenching agent.

In addition, the quenching agent can be a non-denaturing chemical thatis incompatible with enzyme activity. Exemplary chemicals includebuffers and the like that change the pH to a value where the enzymebecomes inactive and thus unable to catalyze the production of theinterfering signal.

Furthermore, the quenching agent can be, for example, an organiccharge-transfer molecule, including 7,7,8,8-tetracyanoquinodimethane(TCNQ), 2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquinodimethane (TFTCNQ),carbon nanotubes, mordant yellow 10 (MY) and4-amino-1,1′-azobenzene-3,4′-disulfonic acid (AB). In preferredembodiments the azobenzene compounds are MY and AB, as they areconsiderably more water-soluble than TCNQ, TFTCNQ and carbon nanotubes.The structure of AB is shown below in:

In some embodiments the quenching agent can be heavy atoms such asiodine which reduces the interference by quenching a fluorescent speciesused to enhance a chemiluminescent signal. In other embodiments thequenching agent can be an organic compound with an absorption spectrumoverlapping the fluorescence emission spectrum of a fluorescent speciesused to enhance a chemiluminescent signal. In some embodiments such aquenching agent is a dark quencher such as a dispersion of carbonparticles (for example, carbon black, charcoal). Carbon can inactivatechemiluminescence by absorbing actives species, and it is also a verygood quenching agent that is substantially incapable of emittingfluorescence.

In yet some embodiments the quenching agent can be an antioxidant, whichcan reduce the interference by disrupting the chemiluminescent reaction.Quenching agents that may be used in some embodiments of the inventioninclude but are not limited to Trolox, butylated hydroxytoluene (BHT),ascorbic acid, citric acid, retinol, carotenoid terpenoids,non-carotenoid terpenoids, phenolic acids and their esters, andbioflavinoids.

In still other embodiments, the quenching agent can be a singlet oxygenquencher, which can reduce the interference by disrupting thechemiluminescent reaction. Some singlet oxygen quenchers include but arenot limited to 1,4 diazabicyclo [2,2,2] octane, thiol containingcompounds such as methionine or cysteine, and carotenoids such aslycopene. In general, the substance used to impregnate or saturate theabsorbent material is preferably highly concentrated, typically in largemolar excess of the assay reagents.

The fluidic device may be manufactured by stamping, thermal bonding,adhesives or, in the case of certain substrates, for example, glass, orsemi-rigid and non-rigid polymeric substrates, a natural adhesionbetween the two components. In some embodiments the fluidic device ismanufactured by ultrasonic or acoustic welding.

The present system also provides a fluidic device that can run a varietyof assays, regardless of the analyte being detected from a bodily fluidsample. A protocol dependent on the identity of the fluidic device maybe transferred from an external device where it can be stored to areader assembly to enable the reader assembly to carry out the specificprotocol on the fluidic device. In preferred embodiments, the fluidicdevice has an identifier (ID) that is detected or read by an identifierdetector described herein. The identifier detector communicates with acommunication assembly via a controller which transmits the identifierto an external device. Where desired, the external device sends aprotocol stored on the external device to the communication assemblybased on the identifier. The protocol to be run on the fluidic devicemay comprise instructions to the controller of the reader assembly toperform the protocol on the fluidic device, including but not limited toa particular assay to be run and a detection method to be performed.Once the assay is performed on the fluidic device, a signal indicativeof an analyte in the bodily fluid sample is generated and detected by adetection assembly. The detected signal may then be communicated to thecommunications assembly, where it can be transmitted to the externaldevice for processing, including without limitation, calculation of theanalyte concentration in the sample.

In some embodiments the identifier may be a bar code identifier with aseries of black and white lines, which can be read by an identifierdetector such as a bar code reader, which are well known. Otheridentifiers could be a series of alphanumerical values, colors, raisedbumps, or any other identifier which can be located on a fluidic deviceand be detected or read by an identifier detector. In some embodimentsthe identifier may comprise a storage or memory device and can transmitinformation to an identification detector. In some embodiments bothtechniques may be used.

Once a bodily fluid sample is provided to a fluidic device, it isinserted in a reader assembly. In some embodiments the fluidic device ispartially inserted manually, and then a mechanical switch in the readerassembly automatically properly positions the fluidic device inside thereader assembly. Any other mechanism known in the art for inserting adisk or cartridge into a device may be used as well. In some embodimentsonly manual insertion may be required.

In some embodiments the reader assembly comprises an identifier detectorfor detecting or reading an identifier on the fluidic device, acontroller for automatically controlling the detection assembly and alsomechanical components of the reader assembly, for example, pumps and/orvalves for controlling or directing fluid through the fluidic device, adetection device for detecting a signal created by an assay run on thefluidic device, and a communication assembly for communicating with anexternal device.

An identifier detector detects an identifier on the fluidic device whichis communicated to a communication assembly. In some embodiments theidentifier detector can be a bar code scanner-like device, reading a barcode on a fluidic device. The identifier detector may also be an LEDthat emits light which can interact with an identifier which reflectslight and is measured by the identifier detector to determine theidentity of a fluidic device.

A reader assembly preferably houses a detection assembly for detecting asignal produced by at least one assay on the fluidic device. Thedetection assembly may be above the fluidic device or at a differentorientation in relation to the fluidic device based on, for example, thetype of assay being performed and the detection mechanism beingemployed.

In preferred embodiments an optical detector is used as the detectiondevice. Non-limiting examples include a photodiode, photomultiplier tube(PMT), photon counting detector, avalanche photo diode, orcharge-coupled device (CCD). In some embodiments a pin diode may beused. In some embodiments a pin diode can be coupled to an amplifier tocreate a detection device with a sensitivity comparable to a PMT. Someassays may generate luminescence as described herein. In someembodiments chemiluminescence is detected. In some embodiments adetection assembly could include a plurality of fiber optic cablesconnected as a bundle to a CCD detector or to a PMT array. The fiberoptic bundle could be constructed of discrete fibers or of many smallfibers fused together to form a solid bundle. Such solid bundles arecommercially available and easily interfaced to CCD detectors.

In some embodiments, the detection system may comprise non-opticaldetectors or sensors for detecting a particular parameter of a patient.Such sensors may include temperature, conductivity, potentiometric, andamperometric, for compounds that are oxidized or reduced, for example,O₂, H₂O₂, and I₂, or oxidizable/reducible organic compounds.

A communication assembly is preferably housed within the reader assemblyand is capable of transmitting and receiving information wirelessly froman external device. Such wireless communication may be bluetooth or RTMtechnology. Various communication methods can be utilized, such as adial-up wired connection with a modem, a direct link such as a T1, ISDN,or cable line. In preferred embodiments a wireless connection isestablished using exemplary wireless networks such as cellular,satellite, or pager networks, GPRS, or a local data transport systemsuch as Ethernet or token ring over a local area network. In someembodiments the information is encrypted before it is transmitted over awireless network. In some embodiments the communication assembly maycontain a wireless infrared communication component for sending andreceiving information.

In some embodiments the communication assembly can have a memory orstorage device, for example localized RAM, in which the informationcollected can be stored. A storage device may be required if informationcan not be transmitted at a given time due to, for example, a temporaryinability to wirelessly connect to a network. The information can beassociated with the fluidic device identifier in the storage device. Insome embodiments the communication assembly can retry sending the storedinformation after a certain amount of time. In some embodiments thememory device can store the information for a period of ten days beforeit is erased.

In preferred embodiments an external device communicates with thecommunication assembly within the reader assembly. An external devicecan wirelessly communicate with a reader assembly, but can alsocommunicate with a third party, including without limitation a patient,medical personnel, clinicians, laboratory personnel, or others in thehealth care industry.

In some embodiments the external device can be a computer system,server, or other electronic device capable of storing information orprocessing information. In some embodiments the external device includesone or more computer systems, servers, or other electronic devicescapable of storing information or processing information. In someembodiments an external device may include a database of patientinformation, for example but not limited to, medical records or patienthistory, clinical trial records, or preclinical trial records. Inpreferred embodiments, an external device stores protocols to be run ona fluidic device which can be transmitted to the communication assemblyof a reader assembly when it has received an identifier indicating whichfluidic device has been inserted in the reader assembly. In someembodiments a protocol can be dependent on a fluidic device identifier.In some embodiments the external device stores more than one protocolfor each fluidic device. In other embodiments patient information on theexternal device includes more than one protocol. In preferredembodiments the external server stores mathematical algorithms toprocess a photon count sent from a communication assembly and in someembodiments to calculate the analyte concentration in a bodily fluidsample.

In some embodiment the external device can include one or more serversas are known in the art and commercially available. Such servers canprovide load balancing, task management, and backup capacity in theevent of failure of one or more of the servers or other components ofthe external device, to improve the availability of the server. A servercan also be implemented on a distributed network of storage andprocessor units, as known in the art, wherein the data processingaccording to the present invention reside on workstations such ascomputers, thereby eliminating the need for a server.

A server can includes a database and system processes. A database canreside, within the server, or it can reside on another server systemthat is accessible to the server. As the information in a database maycontains sensitive information, a security system can be implementedthat prevents unauthorized users from gaining access to the database.

One advantage of the present invention is that information can betransmitted from the external device back to not only the readerassembly, but to other parties or other external devices, for examplewithout limitation, a PDA or cell phone. Such communication can beaccomplished via a wireless network as disclosed herein. In someembodiments a calculated analyte concentration or other patientinformation can be sent to, for example but not limited to, medicalpersonal or the patient.

Methods of Use

The subject apparatus and systems provide an effective means for highthroughput and real-time detection of analytes present in a bodily fluidfrom a subject. The detection methods may be used in a wide variety ofcircumstances including identification and quantification of analytesthat are associated with specific biological processes, physiologicalconditions, disorders or stages of disorders. As such, the subjectapparatus and systems have a broad spectrum of utility in, for example,drug screening, disease diagnosis, phylogenetic classification, parentaland forensic identification, disease onset and recurrence, individualresponse to treatment versus population bases, and monitoring oftherapy. The subject apparatus and systems are also particularly usefulfor advancing preclinical and clinical stage of development oftherapeutics, improving patient compliance, monitoring ADRs associatedwith a prescribed drug, developing individualized medicine, outsourcingblood testing from the central laboratory to the home or on aprescription basis, and monitoring therapeutic agents followingregulatory approval.

Accordingly, in one embodiment, the present invention provides a methodof detecting an analyte in a bodily fluid from a subject. The methodinvolves the steps of a) providing a subject fluidic device or system;b) allowing a portion of said sample to react with assay reagentscontained within said assay assembly to yield a signal indicative of thepresence of said analyte in said sample; and c) detecting said signalgenerated from said analyte collected in said sample of bodily fluid. Inone aspect, the invention may further comprise calibrating themeasurement of said signal generated from said analyte based upon ameasurement by said calibration unit. In another aspect, the method mayfurther comprise the step of quantifying the amount of said analytepresent in said bodily fluid based on the measurement provided by thecalibration unit. The method can be employed to detect a plurality ofanalytes and said fluidic device comprises immunoassay reagents for saidplurality of analytes.

As used herein, the term “subject” or “patient” is used interchangeablyherein, which refers to a vertebrate, preferably a mammal, morepreferably a human. Mammals include, but are not limited to, murines,simians, humans, farm animals, sport animals, and pets.

In some embodiments a sample of bodily fluid can first be provided tothe fluidic device by any of the methods described herein. The fluidicdevice can then be inserted into the reader assembly. An identificationdetector housed within the reader assembly can detect an identifier ofthe fluidic device and communicate the identifier to a communicationassembly, which is preferably housed within the reader assembly. Thecommunication assembly then transmits the identifier to an externaldevice which transmits a protocol to run on the fluidic device based onthe identifier to the communication assembly. In some embodiments thefirst step of the assay is a wash cycle where all the surfaces withinthe fluidic device are wetted using a wash buffer. The fluidic device isthen calibrated using a calibration assembly by running the samereagents as will be used in the assay through the calibration reactionsites, and then a luminescence signal from the reactions sites isdetected by the detection means, and the signal is used in calibratingthe fluidic device. The sample containing the analyte is introduced intothe fluidic channel. The sample may be diluted and further separatedinto plasma or other desired component by a filter. The sample can alsobe separated into plasma by use of magnetic means. The separated samplenow flows through the reaction sites and analytes present therein willbind to reactants bound thereon. The plasma of the sample is thenflushed out of the reaction wells into a waste chamber. Depending on theassay being run, appropriate reagents are directed through the reactionsites to carry out the assay. All the wash buffers and other reagentsused in the various steps; including a calibration step, are collectedin wash tanks. The signal produced in the reaction sites is thendetected by any of the methods described herein.

In some embodiments the method of detecting an analyte in a bodily fluidfrom a subject includes metering a predetermined portion of the sampleto be assayed in the sample collection unit and allowing thepredetermined portion of the sample to react with assay reagentscontained within the assay assembly to yield a signal indicative of thepresence of the analyte in the sample.

In one embodiment, the subject collects a sample of bodily fluid withthe sample dilution syringe. The syringe can be inverted to re-suspendany magnetizable particles contained therein. The sample can enter thesyringe through a capillary tube. In an embodiment measuring an analytein a blood sample, the subject performs a fingerstick and touches theouter end of the glass capillary to the blood so that blood is drawn bycapillary action and fills the capillary with a volume. In preferableembodiments, the sample volume is known. In some embodiments, the samplevolume is in the range of about 5-20 microliters.

The sample dilution syringe is reinserted into device after collectingthe sample. The entire device is then inserted into the reader(instrument). In a preferred embodiment, the rest of the steps of themethod for conducting an assay are performed automatically by a reader,optionally under program control through a wireless link with a server.

The instrument pushes a piston vertically so that the seal of the sampledilution syringe are displaced downwards until fluid connection is madewith a hole in fluid communication with the capillary tube. The sampleand diluent are displaced completely into the well and mixed together bya moving magnetic field provided by the instrument. No liquid proceedsdown the capillary tube as it is sealed at its distal end by a featurein the instrument. The sample collection unit allows for a precisesample dilution to occur because, at least, the sample dilution syringeis adapted to provide a predetermined volume of diluted sample to thesample collection well because of the capillary tube and a precisevolume of diluent is stored in the sample dilution syringe. Because thediluent volume is accurately filled during manufacturing, the sample isdiluted with a high degree of precision.

In an embodiment analyzing a blood sample, the red blood cells in adiluted sample co-agglutinate with the magnetizable particles aided by asolution phase antibody.

In another embodiment, a method and system is provided to obtain aplasma sample substantially free of red blood cells from a blood samplefrom the sample collection unit. When conducting an immunoassay, theanalytes are typically contained in the blood plasma, and the red bloodcells can interfere with a reaction.

The sample is transferred from the sample dilution syringe to a samplecollection well in the device. The instrument contains a method ofoffering a magnetic force to the sample collection well. In the case ofblood analysis, after a short time, the magnetically susceptible mass ofred cells and magnetizable particles are held in the well by themagnetic force. By removing the red blood cells, a blood sample analyzedby the invention is intended to only comprise plasma and diluent. Theplasma and diluent can also be referred to herein as a diluted plasmasample.

The sample cannot proceed from the sample collection well until a sealthe well and a fluidic channel is opened by the instrument. When theseal between the channel and the instrument is opened, the sample movesby capillarity through the fluid channel and vias.

In a preferable embodiment of the invention, the instrument preciselyand accurately controls the temperature of the device. In a furtherembodiment, the temperature is in the range of about 30-40 degreesCelsius.

As the sample flows through the device, it passes through a fluidicchannel with electrodes located within the channel. The conductivity ofthe sample can be measured at this point of the method. The method ofmeasuring the conductivity of a sample is described later herein.

In an embodiment, the sample is a blood sample that has had the redblood cells removed from the sample. The sample has also been dilutedwith a diluent. The resulting diluted plasma sample passes through theassay assembly for analysis.

Before or after the conductivity of the sample has been measured, thesample passes to one or more reaction sites. At the reaction sites, theanalytes bind to their respective capture surfaces. Once the analyteshave bound to the reaction site, the sample is displaced after a knowntime by the liquid reagents in turn flowing in the reverse direction tothat by which the sample entered the channel.

Each assay has its own set of reagents that flow uniquely to theappropriate capture surfaces. Flow is initiated by forcing eachreservoir onto the corresponding needle until the needle completelypierces the lower septum of the vial containing the reagent. Liquidmovement is propelled by plungers in the instrument that engage the topseptum of each reagent reservoir. The plungers push the reagent throughthe needles and into the reagent manifold then into the fluidic channel.Each step for delivering the reagent to the reaction site can be timedprecisely and accurately. After one reagent has been delivered andreacted, it can be displaced by another reagent. The displaced reagentcan then travel into a waste chamber surrounding the reaction site andbe captured by an absorbent material in the waste chamber.

In a preferable embodiment, the final step in the assay is the reactionbetween enzyme captured on the capture surfaces and a chemiluminogenicsubstrate. There is a known relationship between the analyteconcentration and the rate of production of photons. This rate can bemeasured by the instrument.

A variety of assays may be performed on a fluidic device according tothe present invention to detect an analyte of interest in a sample. Awide diversity of labels is available in the art that can be employedfor conducting the subject assays. In some embodiments labels aredetectable by spectroscopic, photochemical, biochemical, immunochemical,or chemical means. For example, useful nucleic acid labels include 32P,35S, fluorescent dyes, electron-dense reagents, enzymes, biotin,dioxigenin, or haptens and proteins for which antisera or monoclonalantibodies are available. A wide variety of labels suitable for labelingbiological components are known and are reported extensively in both thescientific and patent literature, and are generally applicable to thepresent invention for the labeling of biological components. Suitablelabels include radionucleotides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties,bioluminescent labels, or colorimetric labels. Reagents defining assayspecificity optionally include, for example, monoclonal antibodies,polyclonal antibodies, proteins, or other polymers such as affinitymatrices, carbohydrates or lipids. Detection can proceed by any of avariety of known methods, including spectrophotometric or opticaltracking of radioactive, fluorescent, or luminescent markers, or othermethods which track a molecule based upon size, charge or affinity. Adetectable moiety can be of any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of gel electrophoresis, column chromatography, solidsubstrates, spectroscopic techniques, and the like, and in general,labels useful in such methods can be applied to the present invention.Thus, a label includes without limitation any composition detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical thermal, or other chemical means.

In some embodiments the label is coupled directly or indirectly to amolecule to be detected such as a product, substrate, or enzyme,according to methods well known in the art. As indicated above, a widevariety of labels are used, with the choice of label depending on thesensitivity required, ease of conjugation of the compound, stabilityrequirements, available instrumentation, and disposal provisions. Nonradioactive labels are often attached by indirect means. Generally, areceptor specific to the analyte is linked to a signal generatingmoiety. Sometimes the analyte receptor is linked to an adaptor molecule(such as biotin or avidin) and the assay reagent set includes a bindingmoiety (such as a biotinylated reagent or avidin) that binds to theadaptor and to the analyte. The analyte binds to a specific receptor onthe reaction site. A labeled reagent can form a sandwich-like complex inwhich the analyte is in the center. The reagent can also compete withthe analyte for receptors on the reaction site or bind to vacantreceptors on the reaction site not occupied by analyte. The label iseither inherently detectable or covalently bound to a signal system,such as a detectable enzyme, a fluorescent compound, a chemiluminescentcompound, or a chemiluminogenic. A number of ligands and anti-ligandscan be used. Where a ligand has a natural anti-ligand, for example,biotin, thyroxine, digoxigenin, and cortisol, it can be used inconjunction with labeled, anti-ligands. Alternatively, any haptenic orantigenic compound can be used in combination with an antibody.

In some embodiments the label can also be conjugated directly to signalgenerating compounds, for example, by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, oroxidoreductases, particularly peroxidases. Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl,and umbelliferone. Chemiluminescent compounds include dioxetanes,luciferin, and 2,3-dihydrophthalazinediones, such as luminol.

Methods of detecting labels are well known to those of skill in the art.Thus, for example, where the label is radioactive, means for detectioninclude scintillation counting or photographic films as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence by, for example,microscopy, visual inspection, via photographic film, by the use ofelectronic detectors such as digital cameras, charge coupled devices(CCDs) or photomultipliers and phototubes, or other detection device.Similarly, enzymatic labels are detected by providing appropriatesubstrates for the enzyme and detecting the resulting reaction product.Finally, simple colorimetric labels are often detected simply byobserving the color associated with the label. For example, conjugatedgold often appears pink, while various conjugated beads appear the colorof the bead.

In some embodiments the detectable signal may be provided byluminescence sources. “Luminescence” is the term commonly used to referto the emission of light from a substance for any reason other than arise in its temperature. In general, atoms or molecules emit photons ofelectromagnetic energy (for example, light) when then move from an“excited state” to a lower energy state (usually the ground state). Ifexciting cause is a photon, the luminescence process is referred to as“photoluminescence”. If the exciting cause is an electron, theluminescence process is referred to as “electroluminescence”. Morespecifically, electroluminescence results from the direct injection andremoval of electrons to form an electron-hole pair, and subsequentrecombination of the electron-hole pair to emit a photon. Luminescencewhich results from a chemical reaction is usually referred to as“chemiluminescence”. Luminescence produced by a living organism isusually referred to as “bioluminescence”. If photoluminescence is theresult of a spin-allowed transition (for example, a single-singlettransition, triplet-triplet transition), the photoluminescence processis usually referred to as “fluorescence”. Typically, fluorescenceemissions do not persist after the exciting cause is removed as a resultof short-lived excited states which may rapidly relax through suchspin-allowed transitions. If photoluminescence is the result of aspin-forbidden transition (for example, a triplet-singlet transition),the photoluminescence process is usually referred to as“phosphorescence”. Typically, phosphorescence emissions persist longafter the exciting cause is removed as a result of long-lived excitedstates which may relax only through such spin-forbidden transitions. A“luminescent label” may have any one of the above-described properties.

Suitable chemiluminescent sources include a compound which becomeselectronically excited by a chemical reaction and may then emit lightwhich serves as the detectible signal or donates energy to a fluorescentacceptor. A diverse number of families of compounds have been found toprovide chemiluminescence under a variety or conditions. One family ofcompounds is 2,3-dihydro-1,4-phthalazinedione. A frequently usedcompound is luminol, which is a 5-amino compound. Other members of thefamily include the 5-amino-6,7,8-trimethoxy- and thedimethylamino[ca]benz analog. These compounds can be made to luminescewith alkaline hydrogen peroxide or calcium hypochlorite and base.Another family of compounds is the 2,4,5-triphenylimidazoles, withlophine as the common name for the parent product. Chemiluminescentanalogs include para-dimethylamino and -methoxy substituents.Chemiluminescence may also be obtained with oxalates, usually oxalylactive esters, for example, p-nitrophenyl and a peroxide such ashydrogen peroxide, under basic conditions. Other useful chemiluminescentcompounds that are also known include —N-alkyl acridinum esters anddioxetanes. Alternatively, luciferins may be used in conjunction withluciferase or lucigenins to provide bioluminescence.

In some embodiments immunoassays are run on the fluidic device. Whilecompetitive binding assays, which are well known in the art, may be runin some embodiments, in preferred embodiments a two-step method is usedwhich eliminates the need to mix a conjugate and a sample beforeexposing the mixture to an antibody, which may be desirable when verysmall volumes of sample and conjugate are used, as in the fluidic deviceof the present invention. A two-step assay has additional advantagesover the competitive binding assays when use with a fluidic device asdescribed herein. It combines the ease of use and high sensitivity of asandwich (competitive binding) immunoassay with the ability to assaysmall molecules.

In an exemplary two-step assay, the sample containing analyte firstflows over a reaction site containing antibodies. The antibodies bindthe analyte present in the sample. After the sample passes over thesurface, a solution with analyte conjugated to a marker at a highconcentration is passed over the surface. The conjugate saturates any ofthe antibodies that have not yet bound the analyte. Before equilibriumis reached and any displacement of pre-bound unlabelled analyte occurs,the conjugate is washed off. The amount of conjugate bound to thesurface is then measured by the appropriate technique, and the detectedconjugate is inversely proportional to the amount of analyte present inthe sample.

An exemplary measuring technique for a two-step assay is achemiluminescence enzyme immunoassay. As is known in the field, themarker can be a commercially available marker such asdioxitane-phosphate, which is not luminescent but becomes luminescentafter hydrolysis by, for example, alkaline phosphatase. An enzyme suchas alkaline phosphatase is also passed over the substrate to cause themarker to luminesce. In some embodiments the substrate solution issupplemented with enhancing agents such as, without limitation,fluorescein in mixed micelles, soluble polymers, or PVC which create amuch brighter signal than the luminophore alone. Moreover, an alkalinephosphatase conjugate with a higher turnover number than that used inthe commercial assay is employed. This allows signal generation toproceed much more rapidly and a higher overall signal is achieved. Useof a two-step binding assay thus contributes to higher sensitivitycapabilities of the present invention.

Additionally, TOSCA is less sensitive to matrix effects than othermethodologies. This allows one to work with samples that have not beenextensively pre-processed using standard laboratory techniques such as,for example, solid phase extraction and chromatography. Compared tocompetitive binding assay, for all sample preparations (and dilutions),TOSCA has better sensitivity than competitive binding assays.

The term “analytes” according to the present invention includes withoutlimitation drugs, prodrugs, pharmaceutical agents, drug metabolites,biomarkers such as expressed proteins and cell markers, antibodies,serum proteins, cholesterol, polysaccharides, nucleic acids, biologicalanalytes, biomarker, gene, protein, or hormone, or any combinationthereof. At a molecular level, the analytes can be polypeptideglycoprotein, polysaccharide, lipid, nucleic acid, and a combinationthereof.

Of particular interest are biomarkers are associated with a particulardisease or with a specific disease stage. Such analytes include but arenot limited to those associated with autoimmune diseases, obesity,hypertension, diabetes, neuronal and/or muscular degenerative diseases,cardiac diseases, endocrine disorders, any combinations thereof.

Of also interest are biomarkers that are present in varying abundance inone or more of the body tissues including heart, liver, prostate, lung,kidney, bone marrow, blood, skin, bladder, brain, muscles, nerves, andselected tissues that are affected by various disease, such as differenttypes of cancer (malignant or non-metastatic), autoimmune diseases,inflammatory or degenerative diseases.

Also of interest are analytes that are indicative of a microorganism.Exemplary microorganisms include but are not limited to bacterium,virus, fungus and protozoa. Analytes that can be detected by the subjectmethod also include blood-born pathogens selected from a non-limitinggroup that consists of Staphylococcus epidermidis, Escherichia coli,methicillin-resistant Staphylococcus aureus (MSRA), Staphylococcusaureus, Staphylococcus hominis, Enterococcus faecalis, Pseudomonasaeruginosa, Staphylococcus capitis, Staphylococcus warneri, Klebsiellapneumoniae, Haemophilus influnzae, Staphylococcus simulans,Streptococcus pneumoniae and Candida albicans.

Analytes that can be detected by the subject method also encompass avariety of sexually transmitted diseases selected from the following:gonorrhea (Neisseria gorrhoeae), syphilis (Treponena pallidum),chlamydia (Clamydia tracomitis), nongonococcal urethritis (Ureaplasmurealyticum), yeast infection (Candida albicans), chancroid (Haemophilusducreyi), trichomoniasis (Trichomonas vaginalis), genital herpes (HSVtype I & II), HIV I, HIV II and hepatitis A, B, C, G, as well ashepatitis caused by TTV.

Additional analytes that can be detected by the subject methodsencompass a diversity of respiratory pathogens including but not limitedto Pseudomonas aeruginosa, methicillin-resistant Staphlococccus aureus(MSRA), Klebsiella pneumoniae, Haemophilis influenzae, Staphlococcusaureus, Stenotrophomonas maltophilia, Haemophilis parainfluenzae,Escherichia coli, Enterococcus faecalis, Serratia marcescens,Haemophilis parahaemolyticus, Enterococcus cloacae, Candida albicans,Moraxiella catarrhalis, Streptococcus pneumoniae, Citrobacter freundii,Enterococcus faecium, Klebsella oxytoca, Pseudomonas fluorscens,Neiseria meningitidis, Streptococcus pyogenes, Pneumocystis carinii,Klebsella pneumoniae Legionella pneumophila, Mycoplasma pneumoniae, andMycobacterium tuberculosis.

Listed below are additional exemplary markers according to the presentinvention: Theophylline, CRP, CKMB, PSA, Myoglobin, CA125, Progesterone,TxB2,6-keto-PGF-1-alpha, and Theophylline, Estradiol, Lutenizinghormone, High sensitivity CRP, Triglycerides, Tryptase, Low densitylipoprotein Cholesterol, High density lipoprotein Cholesterol,Cholesterol, IGFR.

Exemplary liver markers include without limitation LDH, (LD5), (ALT),Arginase 1 (liver type), Alpha-fetoprotein (AFP), Alkaline phosphatase,Alanine aminotransferase, Lactate dehydrogenase, and Bilirubin.

Exemplary kidney markers include without limitation TNFa Receptor,Cystatin C, Lipocalin-type urinary prostaglandin D, synthatase (LPGbS),Hepatocyte growth factor receptor, Polycystin 2, Polycystin 1,Fibrocystin, Uromodulin, Alanine, aminopeptidase,N-acetyl-B-D-glucosaminidase, Albumin, and Retinol-binding protein(RBP).

Exemplary heart markes include without limitation Troponin I (TnI),Troponin T (TnT), CK, CKMB, Myoglobin, Fatty acid binding protein(FABP), CRP, D-dimer, S-100 protein, BNP, NT-proBNP, PAPP-A,Myeloperoxidase (MPO), Glycogen phosphorylase isoenzyme BB (GPBB),Thrombin Activatable Fibrinolysis Inhibitor (TAFI), Fibrinogen, Ischemiamodified albumin (IMA), Cardiotrophin-1, and MLC-I (Myosin LightChain-I).

Exemplary pancrease markers include without limitation Amylase,Pancreatitis-Assocoated protein (PAP-1), and Regeneratein proteins(REG).

Exemplary muscle tissue markers include without limitation Myostatin.

Exemplary blood markers include without limitation Erythopoeitin (EPO).

Exemplary bone markers include without limitation, Cross-linkedN-telopeptides of bone type I collagen (NTx) Carboxyterminalcross-linking telopeptide of bone collagen, Lysyl-pyridinoline(deoxypyridinoline), Pyridinoline, Tartrate-resistant acid phosphatase,Procollagen type I C propeptide, Procollagen type I N propeptide,Osteocalcin (bone gla-protein), Alkaline phosphatase, Cathepsin K, COMP(Cartillage Oligimeric Matrix Protein), Osteocrin Osteoprotegerin (OPG),RANKL, sRANK, TRAP 5 (TRACP 5), Osteoblast Specific Factor 1 (OSF-1,Pleiotrophin), Soluble cell adhesion molecules, sTfR, sCD4, sCD8, sCD44,and Osteoblast Specific Factor 2 (OSF-2, Periostin).

In some embodiments markers according to the present invention aredisease specific. Exemplary cancer markers include without limitationPSA (total prostate specific antigen), Creatinine, Prostatic acidphosphatase, PSA complexes, Prostrate-specific gene-1, CA 12-5,Carcinoembryonic Antigen (CEA), Alpha feto protein (AFP), hCG (Humanchorionic gonadotropin), Inhibin, CAA Ovarian C1824, CA 27.29, CA 15-3,CAA Breast C1924, Her-2, Pancreatic, CA 19-9, Carcinoembryonic Antigen,CAA pancreatic, Neuron-specific enolase, Angiostatin DcR3 (Soluble decoyreceptor 3), Endostatin, Ep-CAM (MK-1), Free Immunoglobulin Light ChainKappa, Free Immunoglobulin Light Chain Lambda, Herstatin, ChromograninA, Adrenomedullin, Integrin, Epidermal growth factor receptor, Epidermalgrowth factor receptor-Tyrosine kinase, Pro-adrenomedullin N-terminal 20peptide, Vascular endothelial growth factor, Vascular endothelial growthfactor receptor, Stem cell factor receptor, c-kit/KDR, KDR, and Midkine.

Exemplary infectious disease markers include without limitation Viremia,Bacteremia, Sepsis, PMN Elastase, PMN elastase/α1-PI complex, SurfactantProtein D (SP-D), HBVc antigen, HBVs antigen, Anti-HBVc, Anti-HIV,T-supressor cell antigen, T-cell antigen ratio, T-helper cell antigen,Anti-HCV, Pyrogens, p24 antigen, Muramyl-dipeptide.

Exemplary diabetes markers include without limitation C-Peptide,Hemoglobin A1c, Glycated albumin, Advanced glycosylation end products(AGEs), 1,5-anhydroglucitol, Gastric Inhibitory Polypeptide, Glucose,Hemoglobin, ANGPTL3 and 4.

Exemplary inflammation markers include without limitation Rheumatoidfactor (RF), Antinuclear Antibody (ANA), C-reactive protein (CRP), ClaraCell Protein (Uteroglobin).

Exemplary allergy markers include without limitation Total IgE andSpecific IgE.

Exemplary autism markers include without limitation Ceruloplasmin,Metalothioneine, Zinc, Copper, B6, B12, Glutathione, Alkalinephosphatase, and Activation of apo-alkaline phosphatase.

Exemplary coagulation disorders markers include without limitationb-Thromboglobulin, Platelet factor 4, Von Willebrand factor.

In some embodiments a marker may be therapy specific. COX inhibitorsinclude without limitation TxB2 (Cox-1), 6-keto-PGF-1-alpha (Cox 2),11-Dehydro-TxB-1a (Cox-1).

Other markers of the present include without limitation Leptin, Leptinreceptor, and Procalcitonin, Brain S100 protein, Substance P,8-Iso-PGF-2a.

Exemplary geriatric markers include without limitation, Neuron-specificenolase, GFAP, and S100B.

Exemplary markers of nutritional status include without limitationPrealbumin, Albumin, Retinol-binding protein (RBP), Transferrin,Acylation-Stimulating Protein (ASP), Adiponectin, Agouti-Related Protein(AgRP), Angiopoietin-like Protein 4 (ANGPTL4, FIAF), C-peptide, AFABP(Adipocyte Fatty Acid Binding Protein, FABP4) Acylation-StimulatingProtein (ASP), EFABP (Epidermal Fatty Acid Binding Protein, FABP5),Glicentin, Glucagon, Glucagon-Like Peptide-1, Glucagon-Like Peptide-2,Ghrelin, Insulin, Leptin, Leptin Receptor, PYY, RELMs, Resistin, andsTfR (soluble Transferrin Receptor).

Exemplary markers of Lipid metabolism include without limitationApo-lipoproteins (several), Apo-A1, Apo-B, Apo-C-CII, Apo-D, Apo-E.

Exemplary coagulation status markers include without limitation FactorI: Fibrinogen, Factor II: Prothrombin, Factor III: Tissue factor, FactorIV: Calcium, Factor V: Proaccelerin, Factor VI, Factor VII:Proconvertin, Factor VIII: Anti-hemolytic factor, Factor IX: Christmasfactor, Factor X: Stuart-Prower factor, Factor XI: Plasma thromboplastinantecedent, Factor XII: Hageman factor, Factor XIII: Fibrin-stabilizingfactor, Prekallikrein, High-molecular-weight kininogen, Protein C,Protein S, D-dimer, Tissue plasminogen activator, Plasminogen,a2-Antiplasmin, Plasminogen activator inhibitor 1 (PAI1).

Exemplary monoclonal antibodies include those for EGFR, ErbB2, andIGF1R.

Exemplary tyrosine kinase inhibitors include without limitation Ab1,Kit, PDGFR, Src, ErbB2, ErbB 4, EGFR, EphB, VEGFR1-4, PDGFRb, FLt3,FGFR, PKC, Met, Tie2, RAF, and TrkA.

Exemplary Serine/Threoline Kinas Inhibitors include without limitationAKT, Aurora A/B/B, CDK, CDK (pan), CDK1-2, VEGFR2, PDGFRb, CDK4/6,INZEK1-2, mTOR, and PKC-beta.

GPCR targets include without limitation Histamine Receptors, SerotoninReceptors, Angiotensin Receptors, Adrenoreceptors, MuscarinicAcetylcholine Receptors, GnRH Receptors, Dopamine Receptors,Prostaglandin Receptors, and ADP Receptors.

In a separate embodiment, the present invention provides a method ofmonitoring more than one pharmacological parameter useful for assessingefficacy and/or toxicity of a therapeutic agent. The method comprisessubjecting a sample of bodily fluid from a subject administered with thetherapeutic agent to a fluidic device for monitoring said more than onepharmacological parameter, said fluidic device comprising at least onesample collection unit, and an assay assembly comprising reactionreagents; allowing said sample of bodily fluid to react with immunoassayreagents to yield detectable signals indicative of the values of themore than one pharmacological parameter from said sample; and detectingsaid detectable signal generated from said sample of bodily fluid. Wheredesired, the method further involves repeating the steps at a timeinterval prompted by a wireless signal communicated to the subject.

For the purposes of this invention, a “therapeutic agent” is intended toinclude any substances that have therapeutic utility and/or potential.Such substances include but are not limited to biological or chemicalcompounds such as a simple or complex organic or inorganic molecule,peptides, proteins (for example antibodies) or a polynucleotide (forexample anti-sense). A vast array of compounds can be synthesized, forexample polymers, such as polypeptides and polynucleotides, andsynthetic organic compounds based on various core structures, and theseare also included in the term “therapeutic agent”. In addition, variousnatural sources can provide compounds for screening, such as plant oranimal extracts, and the like. It should be understood, although notalways explicitly stated that the agent is used alone or in combinationwith another agent, having the same or different biological activity asthe agents identified by the inventive screen. The agents and methodsalso are intended to be combined with other therapies.

Pharmacodynamic (PD) parameters according to the present inventioninclude without limitation physical parameters such as temperature,heart rate/pulse, blood pressure, and respiratory rate, and biomarkerssuch as proteins, cells, and cell markers. Biomarkers could beindicative of disease or could be a result of the action of a drug.Pharmacokinetic (PK) parameters according to the present inventioninclude without limitation drug and drug metabolite concentration.Identifying and quantifying the PK parameters in real time from a samplevolume is extremely desirable for proper safety and efficacy of drugs.If the drug and metabolite concentrations are outside a desired rangeand/or unexpected metabolites are generated due to an unexpectedreaction to the drug, immediate action may be necessary to ensure thesafety of the patient. Similarly, if any of the pharmacodynamic (PD)parameters fall outside the desired range during a treatment regime,immediate action may have to be taken as well.

In preferred embodiments physical parameter data is stored in orcompared to store profiles of physical parameter data in abioinformatics system which may be on an external device incorporatingpharmacogenomic and pharmacokinetic data into its models for thedetermination of toxicity and dosing. Not only does this generate datafor clinical trials years prior to current processes but also enablesthe elimination of current disparities between apparent efficacy andactual toxicity of drugs through real-time continuous monitoring. Duringthe go/no go decision process in clinical studies, large scalecomparative population studies can be conducted with the data stored onthe database. This compilation of data and real-time monitoring allowsmore patients to enter clinical trials in a safe fashion earlier thancurrently allowed. In another embodiment biomarkers discovered in humantissue studies can be targeted by the device for improved accuracy indetermining drug pathways and efficacy in cancer studies.

In another embodiment, the present invention provides a method ofdetecting at least two distinct analytes of different concentrations ina bodily fluid from a subject comprises providing a fluidic devicecomprising a sample collection unit, an assay assembly, and a pluralityof channels in fluid communication with said sample collection unitand/or said assay assembly; allowing a sample of bodily fluid to reactwith a plurality of reactants contained in said assay assembly to yieldsignals indicative of the concentrations of said at least two analytes;and detecting said signals that are indicative of the presence orabsence of the at least two distinct analytes, wherein said signals aredetectable over a range of 3 orders of magnitude.

Currently, a need exists for the detecting more than one analyte wherethe analytes are present in widely varying concentration range, forexample, one analyte is in the pg/ml concentration and another is in theng/ml concentration. TOSCA described herein has the ability tosimultaneously assay analytes that are present in the same sample in awide concentration range. Another advantage for being able to detectconcentrations of different analytes present in a wide concentrationrange is the ability to relate the ratios of the concentration of theseanalytes to safety and efficacy of multiple drugs administered to apatient. For example, unexpected drug-drug interactions can be a commoncause of adverse drug reactions. A real-time, concurrent measurementtechnique for measuring different analytes would help avoid thepotentially disastrous consequence of adverse drug-drug interactions.

Being able to monitoring the rate of change of an analyte concentrationor PD or PK over a period of time in a single subject, or performingtrend analysis on the concentration, PD, or PK, whether they areconcentrations of drugs or their metabolites, can help preventpotentially dangerous situations. For example, if glucose were theanalyte of interest, the concentration of glucose in a sample at a giventime as well as the rate of change of the glucose concentration over agiven period of time could be highly useful in predicting and avoiding,for example, hypoglycemic events. Such trend analysis has widespreadbeneficial implications in drug dosing regimen. When multiple drugs andtheir metabolites are concerned, the ability to spot a trend and takeproactive measures is often desirable.

Accordingly, the data generated with the use of the subject fluidicdevices and systems can be utilized for performing a trend analysis onthe concentration of an analyte in a subject.

In some embodiments, a method of detecting an analyte in a bodily fluidfrom a subject using an assay transmitted from an external device isprovided. The method comprises providing a fluidic device comprising atleast one sample collection unit and an immunoassay assembly containingimmunoassay reagents; detecting said fluidic device and wirelesslytransmitting an immunoassay protocol to said device; allowing a sampleof bodily fluid to react with immunoassay reagents to yield a detectablesignal indicative of the presence of said analyte using said transmittedimmunoassay protocol; and detecting said detectable signal.

Communication between a reader assembly and an external storage deviceallows for a reader assembly of the present invention to download afluidic device-specific protocol to run on the fluidic device based onthe identity of the fluidic device. This allows a reader assembly to beused interchangeably with any appropriate fluidic device describedherein. In addition, the external device can store a plurality ofprotocols associated with a given fluidic device, and depending on, forexample, a subject's treatment regime or plan, different protocols canbe communicated from the external device to the reader assembly to berun on the fluidic device to detect a variety of analytes. The externaldevice can also store a plurality of protocols associated not only witha fluidic device, but also with a particular subject or subjects, suchthat a protocol can be associated with a subject as well as with afluidic device.

In some embodiments, the present invention provides a business method ofassisting a clinician in providing an individualized medical treatmentcomprises collecting at least one pharmacological parameter from anindividual receiving a medication, said collecting step is effected bysubjecting a sample of bodily fluid to reactants contained in a fluidicdevice, which is provided to said individual to yield a detectablesignal indicative of said at least one pharmacological parameter; andcross referencing with the aid of a computer medical records of saidindividual with the at least one pharmacological parameter of saidindividual, thereby assisting said clinician in providing individualizedmedical treatment.

The present invention allows for automatic quantification of apharmacological parameter of a patient as well as automatic comparisonof the parameter with, for example, the patient's medical records whichmay include a history of the monitored parameter, or medical records ofanother group of subjects. Coupling real-time analyte monitoring with anexternal device which can store data as well as perform any type of dataprocessing or algorithm, for example, provides a device that can assistwith typical patient care which can include, for example, comparingcurrent patient data with past patient data. The present inventiontherefore creates a business method which effectively performs at leastpart of the monitoring of a patient that is currently performed bymedical personnel.

In some embodiments, the present invention provides a business method ofmonitoring a clinical trial of a pharmaceutical agent comprisescollecting at least one pharmacological parameter from a subject in saidclinical trial at a plurality of time intervals, said collecting step iseffected at each time interval by subjecting a sample of bodily fluidfrom said subject to reactants contained in a fluidic device, whereinsaid fluidic device is provided to said subject to yield detectablesignals indicative of the values of said at least one pharmacologicalparameter at a plurality of time intervals; comparing the detectedvalues to a threshold value predetermined for said pharmacologicalparameter; notifying a clinician and/or a sponsor involved in saidclinical trial when a statistically significant discrepancy existsbetween the detected values and the threshold value. Such businessmethods can provide rapidly publishing or generating early reads on newindications, both with respect to sub-patient populations andindications, as well as ameliorating safety concerns. Such methods arealso amenable to conducting a trial involving multiple compounds. Inthis way, multiple compounds can be taken into serially in patientgroups by leveraging this integrated, actionable system.

One advantage of the current invention is that assay results can besubstantially immediately communicated to any third party that maybenefit from obtaining the results. For example, once the analyteconcentration is determined at the external device, it can betransmitted to a patient or medical personnel who may need to takefurther action. The communication step to a third party can be performedwirelessly as described herein, and by transmitting the data to a thirdparty's hand held device, the third party can be notified of the assayresults virtually anytime and anywhere. Thus, in a time-sensitivescenario, a patient may be contacted immediately anywhere if urgentmedical action may be required.

In some embodiments a method of automatically selecting a protocol to berun on a fluidic device comprises providing a fluidic device comprisingan identifier detector and an identifier; detecting said identifier withsaid identifier detector; transferring said identifier to an externaldevice; and selecting a protocol to be run on said fluidic device from aplurality of protocols on said external device associated with saididentifier.

By detecting each fluidic device based on an identifier associated withthe fluidic device after it is inserted in the reader assembly, thesystem of the present invention allows for fluidic device-specificprotocols to be downloaded from an external device and run on thefluidic device. In some embodiments the external device can store aplurality of protocols associated with the fluidic device or associatedwith a particular patient or group of patients. For example, when theidentifier is transmitted to the external device, software on theexternal device can obtain the identifier. Once obtained, software onthe external device, such as a database, can use the identifier toidentify protocols stored in the database associated with theidentifier. If only one protocol is associated with the identifier, forexample, the database can select the protocol and software on theexternal device can then transmit the protocol to the communicationassembly on the reader assembly. The ability to use protocolsspecifically associated with a fluidic device allows for any appropriatefluidic device to be used with a single reader assembly, and thusvirtually any analyte of interest can be detected with a single readerassembly.

In some embodiments multiple protocols may be associated with a singleidentifier. For example, if it is beneficial to detect from the samepatient an analyte once a week, and another analyte twice a week,protocols on the external device associated with the identifier can alsoeach be associated with a different day of the week, so that when theidentifier is detected, the software on the external device can select aspecific protocol that is associated with the day of the week.

In some embodiments a patient may be provided with a plurality offluidic devices to use to detect a variety of analytes. A subject may,for example, use different fluidic devices on different days of theweek. In some embodiments the software on the external deviceassociating the identifier with a protocol may include a process tocompare the current day with the day the fluidic device is to be usedbased on a clinical trial for example. If for example, the two days ofthe week are not identical, the external device can wirelessly sendnotification to the subject using any of the methods described herein orknown in the art to notify them that an incorrect fluidic device is inthe reader assembly and also of the correct fluidic device to use thatday. This example is only illustrative and can easily be extended to,for example, notifying a subject that a fluidic device is not being usedat the correct time of day.

In some embodiments, the present invention provides a method ofobtaining pharmacological data useful for assessing efficacy and/ortoxicity of a pharmaceutical agent from a test animal utilizing thesubject fluidic devices or systems.

When using laboratory animals in preclinical testing of a pharmaceuticalagent, it is often necessary to kill the test subject to extract enoughblood to perform an assay to detect an analyte of interest. This hasboth financial and ethical implications, and as such it may beadvantageous to be able to draw an amount of blood from a test animalsuch that the animal does not need to be killed. In addition, this canalso allow the same test animal to be tested with multiplepharmaceutical agents at different times, thus allowing for a moreeffective preclinical trial. On average, the total blood volume in amouse, for example, is 6-8 ml of blood per 100 gram of body weight. Abenefit of the current invention is that only a very small volume ofblood is required to perform preclinical trials on mice or other smalllaboratory animals. In some embodiment between about 1 microliter andabout 50 microliters are drawn. In an embodiment between about 1microliter and 10 microliters are drawn. In preferred embodiments about5 microliters of blood are drawn.

A further advantage of keeping the test animal alive is evident in apreclinical time course study. When multiple mice, for example, are usedto monitor the levels of an analyte in a test subject's bodily fluidover time, the added variable of using multiple subjects is introducedinto the trial. When, however, a single test animal can be used as itsown control over a course of time, a more accurate and beneficialpreclinical trial can be performed.

In some embodiments a method of automatically monitoring patientcompliance with a medical treatment using the subject fluidic devices orsystems is provided. The method comprises the steps of allowing a sampleof bodily fluid to react with assay reagents in a fluidic device toyield a detectable signal indicative of the presence of an analyte insaid sample; detecting said signal with said fluidic device; comparingsaid signal with a known profile associated with said medical treatmentto determine if said patient is compliant or noncompliant with saidmedical treatment; and notifying a patient of said compliance ornoncompliance.

Noncompliance with a medical treatment, including a clinical trial, canseriously undermine the efficacy of the treatment or trial. As such, insome embodiments the system of the present invention can be used tomonitor patient compliance and notify the patient or other medicalpersonnel of such noncompliance. For example, a patient taking apharmaceutical agent as part of medical treatment plan can take a bodilyfluid sample which is assayed as described herein, but a metaboliteconcentration, for example, detected by the reader assembly may be at anelevated level compared to a known profile that will indicate multipledoses of the pharmaceutical agent have been taken. The patient ormedical personnel may be notified of such noncompliance via any or thewireless methods discussed herein, including without limitationnotification via a handheld device such a PDA or cell phone. Such aknown profile may be located or stored on an external device describedherein.

In some embodiments noncompliance may include taking an improper dose ofa pharmaceutical agent including without limitation multiple doses andno doses, or may include inappropriately mixing pharmaceutical agents.In preferred embodiments a patient is notified substantially immediatelyafter the signal is compared with a known profile.

A patient or subject of a clinical trial may forget to take a bodilyfluid sample as described herein. In some embodiments a method ofalerting a patient to test a sample of bodily fluid using a fluidicdevice as described herein comprises providing a protocol to be run onsaid fluid device, said protocol located on an external device,associated with said patient, and comprising a time and date to testsaid sample of bodily fluid; and notifying patient to test said bodilyfluid on said date and time if said sample has not been tested. In someembodiments a patient can be notified wirelessly as described herein.

A patient may be provided with a fluidic device or devices whenprocuring a prescription of drugs by any common methods, for example, ata pharmacy. Likewise, a clinical trial subject may be provided with suchdevices when starting a clinical trial. The patient or subject's contactinformation, including without limitation cell phone, email address,text messaging address, or other means of wireless communication, may atthat time be entered into the external device and associated with thepatient or subject as described herein, for example, in a database.Software on the external device may include a script or other programthat can detect when a signal generated from a detection device has notyet been sent to the external device, for example at a given time, andthe external device can then send an alert notifying the patient to takea bodily fluid sample.

Methods of Measuring a Plasma Dilution Ratio

In another aspect, the present invention provides a method of measuringplasma concentration in a blood sample. The method comprises the stepsof running a current through the sample and measuring the conductivityof the sample. In a related but separate embodiment, the presentinvention provides a method of calculating an apparent dilution ratioutilized in diluting a blood sample for running a blood test. Thismethod comprises the steps of (a) providing a plasma sample derived froma diluted blood sample; (b) measuring conductivity of the plasma sample;and d) comparing the measured conductivity to a set of predeterminedvalues showing relationship of conductivity values and dilution ratiosthat are employed to dilute a blood sample, thereby calculating theapparent dilution ratio.

The device of the invention can be constructed to detect an analyteutilizing a small volume of bodily fluid. A workable volume of bodilyfluid may range from about 1 microliter to about 500 microliters, fromabout 1 microliter to about 100 microliters, from about 1 microliter toabout 50 microliters, from about 1 microliter to about 30, 20, or even10 microliters.

When such a sample is diluted, the measurement of the analyte in thesample depends on the concentration of the sample in the sample diluentsolution. Traditionally, the sample and the diluent have been carefullymetered and the calculation of the concentration of the sample in adiluted solution was calculated based on the ratio of sample to diluent.In the case of dilution, there can be a target of staying within 2% ofthe metered volumes. This requires a precision of 0.2 microliters for a10 microliter blood sample. In an embodiment where the sample is blood,wherein the whole blood is a somewhat viscous fluid comprising a highvolume fraction (28-58%) of blood cells, it is often more difficult tometer than water or other simple aqueous liquids. It can be difficult toextract and recover blood plasma from such a small sample. Thetheoretical maximum recoverable volume of blood plasma from a 10microliter blood sample with a 50% hematocrit is typically 5microliters.

It can be advantageous to first dilute the blood sample and then removethe red cells to retrieve only a plasma diluent solution (or a plasmadilution sample). The problem introduced by this approach is that thehematocrit (the percentage of red blood cells in blood) has a widevariance, typically from ˜0.28-58% in adults. Therefore, the volume andpercentage of the blood plasma recovered can vary significantly frompatient to patient. To determine the amount of analyte in a bloodsample, the result needs to be concentration per the amount of plasma(or serum) to correspond with clinical laboratory results.

Furthermore, single use devices intended for point-of-care applicationssuffer from the risk that the user will not provide a sample ofsufficient volume and the resulting analytical result will be in error.Even when the device is designed automatically to meter the correctvolume of blood, users can compromise the operation of the device if thedrop of blood obtained is insufficient or the user fails to contact thedevice to the blood drop long enough.

A method to measure the percentage of plasma in a diluted plasma sampleis disclosed. The method solves some of blood processing problemsdescribed herein. Since the measurement is made after all blood cellshave been removed, it can be made independent of the hematocrit. Themethod enables a method of pre-treating a blood sample wherein the bloodis first diluted and then processed for red cell removal. Very precisemetering of the initial blood sample and the diluent is no longer astrict requirement as small errors in dilution will be detected by theplasma percentage measurement. The method enables the reduction of theblood volume required by the device.

The device of the invention comprises a calibration unit, wherein thecalibration unit can comprise two electrodes precisely and accuratelypositioned therein with fixed and precise dimensions. When dilutedplasma travels over the electrodes, the plasma dilution conductivity canbe measured. It is preferred that the temperature of the calibrationunit is controlled and fixed. When an AC voltage is applied to thesample, the impedance is inversely related to the relative concentrationof plasma.

A block diagram of the circuit is shown in FIG. 10. An AC waveform issent by the microprocessor and driven through the sample via theelectrodes. The signal is attenuated by an amount proportional to theconductivity of the sample. The resultant signal is then amplified,reshaped, buffered and sent back to the microprocessor, which thenoutputs a DC voltage that correlates with the conductivity of thesample.

In an embodiment of the device of the invention, the electrodes can beembedded in the straight sample inlet channel before a reaction site. Anexample of a channel adjacent to electrodes is illustrated in FIG. 11.Variations to the conductivity cell can be made to accommodate samplesize and accessibility.

In an embodiment, the material of the electrodes is stainless steel.Other materials that can be used include, but are not limited to,platinum, nickel, and gold. In preferable embodiments, the material ofthe electrodes is inert. In some embodiments, the device of theinvention is intended for single use and the electrode material ischosen based on cost efficiency.

In an example, the spacing of the electrodes is 8 mm and each electrodeis 1.5 mm in diameter. In other embodiments, the spacing can be of arange of about 5-20 mm.

To measure the conductivity of a diluted plasma sample, the fact thatthe conductivity of whole plasma is a tightly controlled parameter canbe taken advantage of. The total conductivity of blood plasma isprimarily attributed to the concentration of sodium and chloride ions,which is relatively constant among most people. The sodium ionconcentration in plasma varies from 136-143 mg/ml (Tietz Textbook ofClinical Chemistry, 2^(nd) ed. p 2206). In cases where a patient'ssodium concentration falls outside this range, it is generallyindicative of very serious organ damage such as kidney failure or heartfailure.

Data from eight samples is shown in FIG. 12. Plasma was collected byspinning down whole blood in a centrifuge. The plasma was then dilutedand the conductivity was measured using a prototype circuit andconductivity cell. The signal for each is referenced to a NaCl controlto account for day to day changes in temperature and the conductivitycell.

The plot in FIG. 12 demonstrates a linear relationship betweenconductivity and relative concentration of plasma in the diluent. Adiluent of low conductivity can be used so that in the diluted plasma,the dominant ionic (current carrying) species derives from the plasma. Acalibration curve is created by measuring the conductivity of knowndilutions of plasma. Then, the dilution of plasma can be calculatedusing the calibration curve.

When measuring the conductivity of sample to obtain the relationship ofthe conductivity and plasma concentration of a diluted sample, a set ofpredetermined values showing relationship of conductivity values andplasma concentrations are plotted and used as a comparison tool.

An apparent dilution ratio refers to the ratio of plasma volume tosample volume as measured by a calibration unit that is configured tomeasure the conductivity of a diluted sample. The apparent dilutionratio reflects the actual dilution factor resulting from adding adiluent to a test sample of bodily fluid. Errors in diluent volume,blood sample volume, and variation in the sample hematocrits results ina discrepancy between the expected dilution ratio and the actual orapparent dilution ratio. To ascertain the concentration of an analyteinitially present in an undiluted fluidic sample, an accurate reading ofthe apparent dilution ratio is desirable.

In an example, the measurable range of the conductivity device wasderived from the following design goals. In certain cases, blood isdiluted 1:10. The conductivity measurement may need to be able tocorrect for errors of up ˜20%. Therefore, the lowest blood dilution inrange is 1:8. In other cases, blood is diluted 1:20. Again, a ˜20% errormust be corrected. The highest blood dilution is then 1:24. Finally, thedetectable range of hematocrit is 28-58%, which makes up most of therest of the blood sample other than the plasma. Using the followingrelationship between plasma dilution and blood dilution:Plasma dilution=Blood dilution*(1/(1−HCT %/100)it can be determined that the measurable range of plasma dilutions needsto be 1:12-1:58. This range can account for the extremes of the designgoals. As this is a fairly large range, two separate gain settings areused on the circuit depending on which dilution is required (1:10 or1:20). This allows maximum resolution in each range of operation. Theeffect of changing the gain setting on the signal and the measurablerange is shown in FIG. 13. The dotted red line indicates the saturationsignal of the detector.

There are several variables that must be optimized when designing theconductivity cell. FIG. 14 demonstrates sample data showing thedependence on electrode spacing and surface area. The output shown is DCvoltage, which, as explained in the description of the block diagram, isrepresentative of the signal attenuation due to the ion carriers in thesample and is thus directly proportional to conductivity. The Figureshows that the closer the electrodes are to one another, the greater themodulation due to changes in salt concentration. However, a secondaryeffect is also observed. When the probes are too close together, theycan begin to act as a single capacitor. Thus, as shown by the data, thebackground signal goes up and the response becomes increasingly noisy.In a preferable embodiment, a probe spacing of 8 mm was selected.

Probes of different diameters were used to expose a different surfacearea to the same volume of fluid. The conductivity of a 40 microlitersample of 15 mM NaCl can be measured. As demonstrated in FIG. 15, adirect relationship is seen between surface area and measured signal,but the slope is fairly shallow, indicating that the signal is onlymoderately sensitive to surface area of the probe.

A more pronounced effect of the relationship of measured signal andvolume is demonstrated in FIG. 16. Smaller volumes result in feweroverall charge carriers and thus a smaller signal.

The measurement of conductivity is temperature sensitive. As thetemperature of the solution increases the viscosity decreases and theions move through the solution more rapidly. This can cause an increasein conductivity of approximately 2% per degree Celsius. This effect isshown in FIG. 16.

One can account for variations in the manufacturing of the conductivitycell or variations in temperature of the conductivity cell by measuringthe conductivity of a reference buffer and normalizing the signal. InFIG. 17, conductivity measurements were taken of a series of NaClsolutions (100 microliter volume). The conductivity of a referencebuffer (TBS) was also taken with each probe. As seen in the first graphthe larger diameter probe yields a higher conductivity for a given NaClsolution. The second graph shows all of the points normalized by thereference buffer conductivity.

The temperature effect can also be corrected by normalizing the signalto a reference solution. The reference solution must be read at the sametemperature as the corresponding samples. In the experiment demonstratedby FIG. 18, a 15 mM NaCl solution was used as a reference. The Figureshows a full set of NaCl dilutions referenced to the 15 mM solution atthree different temperatures.

The ability to normalize the signal is a feature of the device. As shownin the above characterization studies, the signal is sensitive to almostall dimensional parameters of the cell as well as to temperature. Theability to normalize these variations out with a reference buffergreatly improves the reliability of the device.

In another embodiment, the device can measure the conductivity of asample after the red blood cells have been removed. FIG. 19 demonstratesthe effect of red blood cells on the measured conductivity of thesolution. In a device that has a standard dilution of 1:10, the 10× datarepresents no red blood cell removal. The effect on the response isslight but there is a noticeable offset from the response of the puresalt samples. When 90% of the cells are removed (100× blood) the linebasically overlays the “No blood” line. The conductivity measurementsystem is thus robust enough to tolerate a 10% failure in red blood cellremoval.

Another possible failure mode of the blood separation mechanism is celllysis. When the cells are lysed, their contents are released into themedium and the salt concentration of the resulting solution can becomesignificantly altered. This is illustrated by FIG. 20.

A far more pronounced offset in the case of 100% lysed blood (10× lysedblood accounting for the standard 10× dilution) is seen than in theprevious case of unfiltered blood as shown in FIG. 21. However, once 90%of the lysed blood cells are removed (100× lysed blood), the curveoverlays the base case of “No blood.” The conductivity system istherefore also tolerant of up to 10% of the blood cells being lysed withno measurable effect on the signal.

FIG. 22 illustrates an example of clinical data showing the bloodsamples from four patients at varying dilutions and their relation to astandard calibration curve.

EXAMPLE

Cloned human protein-C was diluted into Tris buffer and processed in anembodiment of the device of the present invention. Monoclonal antibodyagainst protein C was adsorbed at 10 ug/mL onto a polystyrene bottompart of a cartridge and then dried. Cartridges were then assembled witha different monoclonal antibody to protein-C labeled with alkalinephosphatase at 25 ng/mL, Tris buffered saline containing BSA (washsolution), and KPL Phosphoglo luminogenic substrate. The reaction siteof the device was incubated with diluted sample and the reagentsincluding a different monoclonal antibody to protein-C labeled withalkaline phosphatase for 10 minutes at room temperature. Followingincubation, the reaction site was washed with 300 microliters of thewash solution, Tris buffered saline containing BSA. Then, the reactionsite was incubated with KPL Phosphoglo luminogenic substrate for 10minutes at room temperature. The assay signal was recorded for 0.5 ms bya photomultiplier in the instrument after the final incubation step. Theresults of the experiment are illustrated in FIG. 23.

1. A method of calculating an apparent dilution ratio utilized indiluting a blood sample for running a blood test, comprising: a)providing a plasma sample derived from a diluted blood sample; b)measuring conductivity of the plasma sample; and c) comparing themeasured conductivity to a set of predetermined values showingrelationship of conductivity values and dilution ratios that areemployed to dilute a blood sample, thereby calculating the apparentdilution ratio utilized in diluting the blood sample.
 2. The method ofclaim 1, wherein the blood sample is diluted prior to running the bloodtest.
 3. The method of claim 1, wherein the plasma sample derived from adiluted blood sample is substantially free of red blood cells.
 4. Themethod of claim 1, wherein conductivity of the plasma sample isinversely proportional to the apparent dilution ratio.
 5. The method ofclaim 1, wherein the measuring of the conductivity of the plasma sampleis performed using at least two electrodes spaced at differentlocations.
 6. The method of claim 5, wherein the electrodes are formedof stainless steel.
 7. The method of claim 5, wherein the electrodes arespaced apart by 5 mm to 20 mm.
 8. The method of claim 1, wherein an ACvoltage is applied to the plasma sample.
 9. The method of claim 8,wherein a relationship between a current induced by the AC voltageapplied and the conductivity of the plasma sample is determined.
 10. Themethod of claim 9, wherein the measured conductivity is amplified,reshaped, buffered, and/or sent to a microprocessor that outputs a DCvoltage correlated to the measured conductivity.
 11. The method of claim1, wherein the blood sample has a volume from about 1 uL to about 500uL.
 12. The method of claim 1, wherein the predetermined values form asubstantially linear calibration curve.
 13. The method of claim 1,wherein the temperature of the plasma sample is fixed.
 14. The method ofclaim 1, wherein the conductivity is measured with a calibration unitcontained in a device that also collects the blood sample.